Topics include membrane structure and function, the molecular bases of hormone action, and the key field of signal transduction. We warmly acknowledge the work of the artists, typesetters, and other individuals unknown to us who participated in the production of the twenty-eighth issue of Harper's Illustrated Biochemistry.
BIOCHEMISTRY & MEDICINE: INTRODUCTION
The Aim of Biochemistry Is to Describe & Explain, in Molecular Terms, All Chemical Processes of Living Cells
A Knowledge of Biochemistry Is Essential to All Life Sciences
A Reciprocal Relationship Between Biochemistry & Medicine Has Stimulated Mutual Advances
His insights were the main foundation for the development of the field of human biochemical genetics. Knowledge of the biochemical molecules shown in the upper part of the diagram has clarified our understanding of the diseases shown in the lower half – and conversely, analyzes of the diseases shown below have shed light on many areas of biochemistry.
NORMAL BIOCHEMICAL PROCESSES ARE THE BASIS OF HEALTH
Recent efforts to understand the basis of the genetic disease known as familial hypercholesterolemia, which results in severe atherosclerosis at an early age, have led to dramatic advances in the understanding of cell receptors and of mechanisms for the uptake of cholesterol into cells. As long as medical treatment is firmly rooted in knowledge of biochemistry and other basic sciences, medical practice will have a rational basis that can be adapted to accommodate new knowledge.
Biochemical Research Has Impact on Nutrition & Preventive Medicine
Most & Perhaps All Diseases Have a Biochemical Basis
Use of the plasma enzyme alanine aminotransferase (ALT) to monitor the progression of infectious hepatitis. Use of measurement of blood carcinoembryonic antigen (CEA) in certain patients treated for cancer of the colon.
Impact of the Human Genome Project (HGP) on Biochemistry, Biology, & Medicine
Some of the major challenges facing medicine and related health sciences are also very briefly outlined at the end of Chapter 54. All of the above has made the present a very exciting one to study or be directly involved in biology and medicine.
SUMMARY
Consulting this extensive collection of diseases and other relevant articles – specific proteins, enzymes, etc. – will greatly enhance the reader's knowledge and understanding of the various topics referenced and discussed in this text. McGraw-Hill, 2001 (This text is now available online and updated as The Online Metabolic & Molecular Bases of Inherited Disease at www.ommbid.com. A subscription is required, although access may be available through university and hospital libraries and other sources) .
GLOSSARY
Glycomics is the systematic study of the structures and functions of glycomes (eg, the human glycome; see Chapter 47). Proteomics is the systematic study of proteome structures and functions, including variations in health and disease (see Chapter 4).
BIOMEDICAL IMPORTANCE
WATER IS AN IDEAL BIOLOGIC SOLVENT Water Molecules Form Dipoles
As described quantitatively by Coulomb's law, the strength of interaction F between oppositely charged particles is inversely proportional to the dielectric constant of the surrounding medium. Its strong dipole and high dielectric constant enable water to dissolve large amounts of charged compounds such as salts.
Water Molecules Form Hydrogen Bonds
For example, the oxygen atoms of aldehydes, ketones, and amides provide lone pairs of electrons that can serve as hydrogen acceptors. Alcohols and amines can serve as hydrogen acceptors and as donors of unprotected hydrogen atoms to form hydrogen bonds (Figure 2–3).
INTERACTION WITH WATER INFLUENCES THE STRUCTURE OF BIOMOLECULES
Covalent & Noncovalent Bonds Stabilize Biologic Molecules
Biomolecules Fold to Position Polar & Charged Groups on Their Surfaces
Hydrophobic Interactions
Thus, non-polar molecules tend to form droplets to minimize the exposed surface area and reduce the number of water molecules affected. Likewise, in the aqueous environment of the living cell, the hydrophobic portions of biopolymers tend to be buried within the structure of the molecule, or within a lipid bilayer, minimizing contact with water.
Electrostatic Interactions
The maximum formation of multiple hydrogen bonds can only be maintained by increasing the order of neighboring water molecules, with an accompanying decrease in entropy.
Multiple Forces Stabilize Biomolecules
WATER IS AN EXCELLENT NUCLEOPHILE
While hydrolysis is a thermodynamically favored reaction, the amide and phosphoester bonds of polypeptides and oligonucleotides are stable in the aqueous environment of the cell. Proteases catalyze the hydrolysis of proteins into their amino acids, while nucleases catalyze the hydrolysis of the phosphoester bonds in DNA and RNA.
Many Metabolic Reactions Involve Group Transfer
In the cell, protein catalysts called enzymes speed up the rate of hydrolytic reactions when needed. Careful control of the activities of these enzymes is required to ensure that they act only on the correct target molecules at the appropriate time.
Water Molecules Exhibit a Slight But Important Tendency to Dissociate
The molar concentration of water, 55.56 mol/L, is too high to be significantly affected by dissociation. This constant can therefore be incorporated into the dissociation constant K to provide a useful new constant K w called the ion product for water.
PH IS THE NEGATIVE LOG OF THE HYDROGEN ION CONCENTRATION
Since pH is determined by the total [H+] (and pOH by the total [OH–]), both sources must be considered. Since weak electrolytes dissociate only slightly in solution, we must use the dissociation constant to calculate the concentration of [H+] (or [OH–]) produced by a given molarity of a weak acid (or base) before calculating total [H+ ] (or total [OH–]) and subsequent pH.
Functional Groups that Are Weak Acids Have Great Physiologic Significance
Knowledge of the dissociation of weak acids and bases is thus fundamental to understanding the influence of intracellular pH on biological structure and activity. The relative strengths of bases are expressed in terms of the pKa of their conjugate acids.
The Henderson–Hasselbalch Equation Describes the Behavior of Weak Acids & Buffers
Since –log Ka is defined as pKa, and –log [H+] defines pH, the equation can be rewritten as The PKa for an acid can be determined by adding 0.5 equivalents of alkali per equivalent of acid.
Solutions of Weak Acids & Their Salts Buffer Changes in pH
Figure 2–4 also illustrates the net charge of one molecule of acid as a function of pH. Considering the net charge of macromolecules as a function of pH provides the basis for separation techniques such as ion exchange chromatography and electrophoresis.
Acid Strength Depends on Molecular Structure
A solution resists pH changes most effectively at pH values close to the pKa. 1Note: The values in the table are the pKa values (–log dissociation constant) of selected monoprotic, diprotic and triprotic acids.
L-amino acids and their derivatives participate in such diverse cellular functions as nerve transmission and the biosynthesis of porphyrins, purines, pyrimidines, and urea. While proteins contain only L-amino acids, microorganisms produce peptides containing both D- and L-amino acids.
PROPERTIES OF AMINO ACIDS
Short polymers of amino acids called peptides play prominent roles in the neuroendocrine system as hormones, hormone-releasing factors, neuromodulators or neurotransmitters. Humans and other higher animals lack the ability to synthesize 10 of the 20 common L - amino acids in amounts sufficient to support infant growth or to maintain adult health.
Several of these peptides are of therapeutic value, including the antibiotics bacitracin and gramicidin A and the antitumor agent bleomycin.
Amino Acids May Have Positive, Negative, or Zero Net Charge
The net charge on an amino acid - the algebraic sum of all the positively and negatively charged groups present - depends on the pK a values of its functional groups and on the pH of the surrounding medium. Changing the charge on amino acids and their derivatives by varying pH facilitates the physical separation of amino acids, peptides and proteins (see Chapter 4).
At Its Isoelectric pH (pI), an Amino Acid Bears No Net Charge
Similar considerations apply to all polyprotic acids (eg proteins), regardless of the number of dissociating groups present. For example, electrophoresis at pH 7.0 will separate two molecules with pI values of 6.0 and 8.0, because at pH 7.0 the molecule with a pI of 6.0 will have a net positive charge, and that with a pI of 8.0, a net negative charge.
Similar considerations apply to understanding chromatographic separations on ionic supports such as diethylaminoethyl (DEAE) cellulose (see Chapter 4).
The Solubility of Amino Acids Reflects Their Ionic Character
Because it absorbs ultraviolet light about ten times more efficiently than phenylalanine or tyrosine, tryptophan is the major contributor to the ability of most proteins to absorb light in the 280 nm region.
THE -R GROUPS DETERMINE THE PROPERTIES OF AMINO ACIDS
FUNCTIONAL GROUPS DICTATE THE CHEMICAL REACTIONS OF AMINO ACIDS
Amino Acid Sequence Determines Primary Structure
Peptide Structures Are Easy to Draw
Add the main chain atoms, which appear in the repeating order: -nitrogen, -carbon, carbonyl carbon. Now add a hydrogen atom to each -carbon and to each peptide nitrogen, and an oxygen atom to the carbonyl carbon.
Some Peptides Contain Unusual Amino Acids
Peptides Are Polyelectrolytes
The Peptide Bond Has Partial Double-Bond Character
Free rotation can occur around the bonds connecting the -carbon to the -nitrogen and the -carbonyl carbon (brown arrows). The extended polypeptide chain is therefore a semi-rigid structure with two-thirds of the backbone atoms in a fixed planar relationship to each other.
Noncovalent Forces Constrain Peptide Conformations
ANALYSIS OF THE AMINO ACID CONTENT OF BIOLOGIC MATERIALS
Of the biochemical reactions of amino acids, the most important is the formation of peptide bonds. Peptides are named according to the number of amino acid residues present and as derivatives of the carboxyl terminal residue.
PROTEINS & PEPTIDES MUST BE PURIFIED PRIOR TO ANALYSIS
For example, subsequent covalent modification can attach a fatty acid molecule (yellow) for (5) translocation of the modified protein to a membrane.
Column Chromatography
Partition Chromatography
Size Exclusion Chromatography
Absorption Chromatography
Proteins are then released sequentially by disrupting the forces that stabilize the protein-stationary phase complex, mostly using a gradient of increasing salt concentration. The composition of the mobile phase is gradually changed so that molecules are selectively released in descending order of their affinity for the stationary phase.
Ion Exchange Chromatography
For absorption chromatography, the protein mixture is applied to a column under conditions where the protein of interest associates with the stationary phase so closely that its partition coefficient is essentially the same.
Hydrophobic Interaction Chromatography
Affinity Chromatography
Peptides Are Purified by Reversed-Phase High-Pressure Chromatography
High-pressure liquid chromatography (HPLC) uses incompressible silica or alumina microspheres as the stationary phase and pressures up to several thousand psi. Peptide mixtures are eluted using a water-miscible organic solvent gradient such as acetonitrile or methanol.
Protein Purity Is Assessed by Polyacrylamide Gel Electrophoresis (PAGE)
Isoelectric Focusing (IEF)
The IEF gel was then placed horizontally on top of an SDS gel, and the proteins were then further resolved by SDS-PAGE. Note the greatly improved resolution of distinct polypeptides compared to conventional SDS-PAGE gel (Figure 4-5).
SANGER WAS THE FIRST TO DETERMINE THE SEQUENCE OF A POLYPEPTIDE
THE EDMAN REACTION ENABLES PEPTIDES & PROTEINS TO BE SEQUENCED
MOLECULAR BIOLOGY REVOLUTIONIZED THE DETERMINATION OF PRIMARY STRUCTURE
MASS SPECTROMETRY DETECTS COVALENT MODIFICATIONS
As the liquid droplet exits the sample chamber, the solvent rapidly disperses, leaving the macromolecule suspended in the gaseous phase. In the sample chamber, the mixture is excited by means of a laser, causing the surrounding matrix to disperse into the vapor phase so quickly that heating of embedded peptides or proteins is avoided (Figure 4-9).
Tandem Mass Spectrometry
Tandem Mass Spectrometry Can Detect Metabolic Abnormalities
GENOMICS ENABLES PROTEINS TO BE IDENTIFIED FROM SMALL AMOUNTS OF SEQUENCE DATA
PROTEOMICS & THE PROTEOME
The Goal of Proteomics Is to Identify the Entire Complement of Proteins Elaborated by a Cell Under Diverse Conditions
Since the human body contains thousands of cell types, each containing thousands of proteins, the proteome—the set of all proteins expressed by an individual cell at a given time—represents a moving target of formidable dimensions.
Two-Dimensional Electrophoresis & Gene Array Chips Are Used to Survey Protein Expression
Bioinformatics Assists Identification of Protein Functions
Genomics—the analysis of the entire oligonucleotide sequence of an organism's complete genetic material—provided further improvements. Austin CP: The impact of the completed human genome sequence on the development of new therapeutics for human disease.
CONFORMATION VERSUS CONFIGURATION
PROTEINS WERE INITIALLY CLASSIFIED BY THEIR GROSS CHARACTERISTICS
PROTEINS ARE CONSTRUCTED USING MODULAR PRINCIPLES
THE FOUR ORDERS OF PROTEIN STRUCTURE
SECONDARY STRUCTURE
Peptide Bonds Restrict Possible Secondary Conformations
The dots represent permitted combinations, and the spaces prohibited combinations, of phi and psi angles. Regions of ordered secondary structure arise when a series of aminoacyl residues adopt similar phi and psi angles.
The Alpha Helix
The stability of a helix derives mainly from hydrogen bonds that are formed between the oxygen of the peptide bond carbonyl and the hydrogen atom of the peptide bond nitrogen from the fourth residue. Many helices have predominantly hydrophobic R groups on one side of the axis of the helix and.
The Beta Sheet
Both configurations allow the maximum number of hydrogen bonds between segments or strands of the sheet. The color of the polypeptide chain is graded along the visible spectrum from purple (N-terminal) to tan (C-terminal).
Loops & Bends
Tertiary & Quaternary Structure
The color of the polypeptide chain is graded along the visible spectrum from blue (N-terminal) to orange (C-terminal). Note how the N-terminal part of the polypeptide forms a continuous domain rich in sheets that binds ADP.
MULTIPLE FACTORS STABILIZE TERTIARY & QUATERNARY STRUCTURE
Homodimers contain two copies of the same polypeptide chain, while in a heterodimer the polypeptides differ. In an even simpler representation, the line segments connecting the carbons indicate the path of the polypeptide backbone.
THREE-DIMENSIONAL STRUCTURE IS DETERMINED BY X-RAY CRYSTALLOGRAPHY OR BY NMR SPECTROSCOPY
Ray Crystallography
Ribbon diagrams (Figures 5–6 and 5–8) trace the conformation of the polypeptide backbone, with cylinders and arrows indicating regions of the helix and sheet, respectively. Before irradiation, an atom with a characteristic X-ray "signature" is introduced into a crystal at known positions in the protein's primary structure.
Crystallography
Expression uses a bacterial host that is exotrophic for methionine biosynthesis and a defined medium in which selenomethionine replaces methionine. Finally, the results of the phasing and Fourier summations provide an electron density profile or three-dimensional map of how the atoms are connected or related to each other.
Nuclear Magnetic Resonance Spectroscopy
Alternatively, if the unknown structure is similar to one already solved, molecular replacement on an existing model provides an attractive way to phase the data without the use of heavy atoms. This not only avoids the need to form crystals (a particular advantage when it is difficult to crystallize membrane proteins), it allows real-time observation of the.
Molecular Modeling
PROTEIN FOLDING
The Native Conformation of a Protein Is Thermodynamically Favored
Folding Is Modular
In the second stage, the hydrophobic regions are separated into the interior of the protein away from the solvent, forming a "molten sphere", a partially coiled polypeptide in which the secondary structure modules are rearranged until the mature conformation of the protein is reached. There is considerable flexibility in the ways and order in which secondary structure elements can be rearranged.
Auxiliary Proteins Assist Folding
Stacking is now reduced to choosing the appropriate arrangement of this relatively small number of secondary structural elements. In general, each secondary or super-secondary structure element facilitates proper folding by directing the folding process toward the native conformation and away from unproductive alternatives.
Chaperones
Protein Disulfide Isomerase
Proline-cis, trans-Isomerase
Isomerization of the N-1 prolyl peptide bond from a cis to a trans configuration relative to the polypeptide backbone.
Folding Is a Dynamic Process
PERTURBATION OF PROTEIN CONFORMATION MAY HAVE PATHOLOGIC CONSEQUENCES
Prions
Alzheimer's Disease
Beta-Thalassemias
COLLAGEN ILLUSTRATES THE ROLE OF POSTTRANSLATIONAL PROCESSING IN PROTEIN MATURATION
Protein Maturation Often Involves Making & Breaking Covalent Bonds
Collagen Is a Fibrous Protein
Collagen Forms a Unique Triple Helix
Collagen Is Synthesized as a Larger Precursor
The central part of the precursor polypeptide then associates with other molecules to form the. The aldehydes can either undergo an aldol condensation to form a C==C double bond, or form a Schiff base (eneimine) with the -amino group of an unmodified lysyl moiety, which is then reduced to form a single C-N bond to shape.
Nutritional & Genetic Disorders Can Impair Collagen Maturation
Stoddard BL et al: Millisecond Laue structures of an enzyme-product complex using photocatalyzed substrate analogs. Cyanide and carbon monoxide kill because they disrupt the physiological function of heme proteins, namely cytochrome oxidase and hemoglobin.
HEME & FERROUS IRON CONFER THE ABILITY TO STORE & TO TRANSPORT OXYGEN
The fifth and sixth coordination positions of Fe2+ are directed perpendicular to - and directly above and below - the plane of the heme ring. Note the nature of the methyl (blue), vinyl (green), and propionate (orange) substituent groups on the carbons of the pyrrole rings, the central iron atom (red), and the location of the polar side of the heme ring (about 7 o'clock) facing with the surface of the myoglobin molecule.
Myoglobin Is Rich in Helix
The color of the polypeptide chain is graded along the visible spectrum from blue (N-terminal) to brown (C-terminal).
Histidines F8 & E7 Perform Unique Roles in Oxygen Binding
The Iron Moves Toward the Plane of the Heme When Oxygen Is Bound
Apomyoglobin Provides a Hindered Environment for Heme Iron
When CO binds to an isolated heme, all three atoms (Fe, C, and O) lie perpendicular to the plane of the heme. The distal E7 histidine hinders CO binding at the desired (90-degree) angle to the plane of the heme ring.
THE OXYGEN DISSOCIATION CURVES FOR MYOGLOBIN & HEMOGLOBIN SUIT THEIR PHYSIOLOGIC ROLES
Binding at a less favorable angle reduces the heme-CO bond strength to about 200 times the heme-O2 bond strength (Figure 6-3, right), at which stage the large excess of O2 over the normally present CO dominates. Oxygen and carbon monoxide (CO) binding angles to the heme iron of myoglobin.
THE ALLOSTERIC PROPERTIES OF HEMOGLOBINS RESULT FROM THEIR QUATERNARY STRUCTURES
Arterial oxygen tension is approximately 100 mm Hg; mixed venous oxygen tension is approximately 40 mm Hg; capillary (active muscle) oxygen tension is about 20 mm Hg; and the minimum oxygen tension required for cytochrome oxidase is about 5 mm Hg.
Hemoglobin Is Tetrameric
The two subunits are colored in the darker shades of green and blue, the two subunits in the lighter shades of green and blue, and the heme prosthetic groups in red.
Myoglobin & the Subunits of Hemoglobin Share Almost Identical Secondary and Tertiary Structures
Oxygenation of Hemoglobin Triggers Conformational Changes in the Apoprotein
P 50 Expresses the Relative Affinities of Different Hemoglobins for Oxygen
Oxygenation of Hemoglobin Is Accompanied by Large Conformational Changes
During the transition from the T form to the R form of hemoglobin, the 2 2 pairs of subunits (green) rotate 15 degrees relative to the pair of 1 1 subunits (yellow). Fully oxygenated molecules in the T structure and fully deoxygenated molecules in the R structure are not shown because they are unstable.
Carbon dioxide generated in peripheral tissues combines with water to form carbonic acid, which dissociates into protons and bicarbonate ions. In the lungs, hemoglobin's uptake of oxygen releases protons, which combine with bicarbonate to form carbonic acid, which, when dehydrated by carbonic anhydrase, becomes carbon dioxide, which is then exhaled.
Protons Arise from Rupture of Salt Bridges When O 2 Binds
2,3-Bisphosphoglycerate (BPG) Stabilizes the T Structure of Hemoglobin
BPG therefore stabilizes deoxygenated (T-state) hemoglobin by forming additional salt bridges that must be broken before conversion to the R-state. The lower stabilization conferred by BPG to the T state accounts for HbF having a higher affinity for O2 than HbA.
Adaptation to High Altitude
The hemoglobin tetramer binds one molecule of BPG in the central cavity formed by its four subunits (Figure 6-4). However, the space between the H-helices of the chains lining the cavity is sufficiently wide to accommodate BPG only when hemoglobin is in the T state.
NUMEROUS MUTATIONS AFFECTING HUMAN HEMOGLOBINS HAVE BEEN IDENTIFIED
Methemoglobin & Hemoglobin M
Hemoglobin S
BIOMEDICAL IMPLICATIONS Myoglobinuria
Anemias
Thalassemias
Glycated Hemoglobin (HbA 1c )
In: The Metabolic and Molecular Bases of Inherited Disease, 8th ed., Scriver CR et al (editors). Deficiencies in the quantity or catalytic activity of key enzymes can result from genetic defects, nutritional deficiencies, or toxins.
ENZYMES ARE EFFECTIVE & HIGHLY SPECIFIC CATALYSTS
The ability to test the activity of specific enzymes in blood, other tissue fluids, or cell extracts helps in the diagnosis and prognosis of disease. The absolute stereospecificity of enzymes is particularly important for use as soluble or immobilized catalysts for specific reactions in the synthesis of a drug or antibiotic.
ENZYMES ARE CLASSIFIED BY REACTION TYPE
Figure 7-1 illustrates why enzyme-catalyzed reduction of the nonchiral substrate pyruvate produces L-lactate—rather than a racemic mixture of D- and L-lactate. Despite the clarity of the IUB system, the names are long and relatively cumbersome, so we generally continue to refer to enzymes by their traditional, albeit sometimes ambiguous, names.
PROSTHETIC GROUPS, COFACTORS, & COENZYMES PLAY IMPORTANT ROLES IN CATALYSIS
Prosthetic Groups Are Tightly Integrated Into an Enzyme's Structure
Cofactors Associate Reversibly with Enzymes or Substrates
Coenzymes Serve as Substrate Shuttles
Many Coenzymes, Cofactors & Prosthetic Groups Are Derivatives of B Vitamins
Nicotinamide is a component of the redox coenzymes NAD and NADP, while riboflavin is a component of the redox coenzymes FMN and FAD.
CATALYSIS OCCURS AT THE ACTIVE SITE
As the name suggests, the active site is much more than just a recognition site for binding substrates. Two-dimensional representation of a dipeptide substrate, glycyl-tyrosine, bound within the active site of carboxypeptidase A.
ENZYMES EMPLOY MULTIPLE MECHANISMS TO FACILITATE CATALYSIS
In most enzymes, the active site takes the form of a cleft or pocket on the enzyme's surface (Figures 5–6 & 5–8) or, for some multimeric enzymes, at the interface between subunits. Within the active site, substrate molecules are aligned with and optimally oriented to the functional groups of peptidylaminoacyl residues, cofactors, and prosthetic groups responsible for catalyzing their chemical transformation into products (Figure 7-3).
Catalysis by Proximity
Acid-Base Catalysis
The ionizable functional groups of aminoacyl side chains and (where present) of prosthetic groups contribute to catalysis by acting as acids or bases. In specific acid or specific base catalysis, the reaction rate is sensitive to changes in the concentration of protons, but independent of the concentrations of other acids (proton donors) or bases (proton acceptors) present in the solution or at the active site.
Catalysis by Strain
Reactions whose rate is responsive to all the acids or bases present are said to be subject to general acid or general base catalysis.
Covalent Catalysis
SUBSTRATES INDUCE CONFORMATIONAL CHANGES IN ENZYMES
The induced fit model has been amply validated by biophysical studies of enzyme motion during substrate binding. A–B substrate binding induces a conformational change in the enzyme that aligns the catalytic residues involved in catalysis and strains the bond between A and B, facilitating its cleavage.
HIV PROTEASE ILLUSTRATES ACID-BASE CATALYSIS
Aspartate Y acts as an acid to facilitate degradation of the tetrahedral intermediate and release of the split products by donating a proton to the newly formed amino group. Subsequent shuttling of the proton on Asp X to Asp Y restores the protease to its native state.
CHYMOTRYPSIN & FRUCTOSE-2,6-BISPHOSPHATASE ILLUSTRATE COVALENT CATALYSIS
Chymotrypsin
The portion of the original peptide with a free amino group then leaves the active site and is replaced by a water molecule. Release of the amino-terminal peptide is facilitated by donation of a proton to the newly formed amino group by His 57 of the charge-relay system, yielding an acyl-Ser 195 intermediate.
Fructose-2,6-Bisphosphatase
The charge relay system donates a proton to Ser 195, facilitating cleavage of the tetrahedral intermediate to release the carboxyl-terminal peptide.
CATALYTIC RESIDUES ARE HIGHLY CONSERVED
ISOZYMES ARE DISTINCT ENZYME FORMS THAT CATALYZE THE SAME REACTION
Isoenzymes can exhibit subtle differences in properties, such as sensitivity to certain regulatory factors (Chapter 9) or substrate affinity (eg, hexokinase and glucokinase), that tailor them to specific tissues or circumstances.
THE CATALYTIC ACTIVITY OF ENZYMES FACILITATES THEIR DETECTION
Single-Molecule Enzymology
Screening
Enzyme-Linked Immunoassays
NAD(P) + -Dependent Dehydrogenases Are Assayed Spectrophotometrically
Many Enzymes Are Assayed by Coupling to a Dehydrogenase
The production of glucose-6-phosphate by hexokinase is coupled to the oxidation of this product by glucose-6-phosphate dehydrogenase in the presence of added enzyme and NADP+. When excess glucose-6-phosphate dehydrogenase is present, the rate of NADPH formation, which can be measured at 340 nm, depends on the rate of glucose-6-phosphate formation by hexokinase.
THE ANALYSIS OF CERTAIN ENZYMES AIDS DIAGNOSIS
Enzymes Assist Diagnosis of Myocardial Infarction
Immunological measurements of plasma levels of cardiac troponins I and T provide sensitive and specific indicators of myocardial damage. Enzymes are increasingly being used as tools to treat injury and disease.
ENZYMES FACILITATE DIAGNOSIS OF GENETIC AND INFECTIOUS DISEASES
RECOMBINANT DNA PROVIDES AN IMPORTANT TOOL FOR STUDYING ENZYMES
Recombinant Fusion Proteins Are Purified by Affinity Chromatography
Site-Directed Mutagenesis Provides Mechanistic Insights
Enzyme kinetics is the field of biochemistry concerned with the quantitative measurement of the rates of enzyme-catalyzed reactions and the systematic study of factors that influence these rates. Together with site-directed mutagenesis and other techniques that investigate protein structure, kinetic analyzes can reveal details of the catalytic mechanism of a particular enzyme.
CHEMICAL REACTIONS ARE DESCRIBED USING BALANCED EQUATIONS
Kinetic analysis can reveal the number and sequence of individual steps by which enzymes transform substrates into products. The rapid removal of product P or Q therefore effectively rules out the occurrence of the reverse reaction, making equation (2) functionally irreversible under physiological conditions.
CHANGES IN FREE ENERGY DETERMINE THE DIRECTION &
EQUILIBRIUM STATE OF CHEMICAL REACTIONS
G0 is independent of the mechanism of the reaction and therefore does not provide any information regarding the rate of reactions.
THE RATES OF REACTIONS ARE DETERMINED BY THEIR ACTIVATION ENERGY
Reactions Proceed Via Transition States
Note how the geometry of the phosphoryl group changes between the transition state and the substrate or product. Reaction (7) can be thought of as consisting of two "part reactions", the first corresponding to the formation (F) and the second to the subsequent decomposition (D) of the transition state intermediate.
G F Defines the Activation Energy
Notice how the three oxygen atoms connected by the triangular lines and the phosphorus atom of the phosphoryl group form a pyramid. For these reactions, the overall G represents the sum of all the free energy changes associated with the.
NUMEROUS FACTORS AFFECT THE REACTION RATE
Middle: As E approaches the L-phosphate, a new bond begins to form between E and the phosphate group (dashed line) as the bond of L to the phosphate group weakens. Note how the phosphorus and three oxygen atoms occupying the four corners of the pyramid in the substrate and product become coplanar in the transition state, as highlighted by the triangle.
Temperature
Reactant Concentration
Therefore, while the overall order of the reaction is two, it is said to be first order with respect to A and first order with respect to B. Under these pseudo-first-order conditions, the concentration of the reactant(s) remains virtually fixed constant.
THE KINETICS OF ENZYMATIC CATALYSIS
Enzymes Lower the Activation Energy Barrier for a Reaction
Catalysis by enzymes that proceeds via a unique reaction mechanism usually occurs when the transition state mediator forms a covalent bond with the enzyme (covalent catalysis). The catalytic mechanism of the serine protease chymotrypsin (see Figure 7-7) illustrates how an enzyme uses covalent catalysis to provide a unique reaction pathway.
ENZYMES DO NOT AFFECT K EQ
MULTIPLE FACTORS AFFECT THE RATES OF ENZYME-CATALYZED REACTIONS
The Q10, or temperature coefficient, is the factor by which the speed of a biological process increases with a temperature increase of 10°C. For the temperatures at which enzymes are stable, the rates of most biological processes typically double with a 10°C increase in temperature (Q1 0 = 2).
Hydrogen Ion Concentration
Changes in the rate of enzyme-catalyzed reactions that accompany a rise or fall in body temperature constitute a prominent survival feature of "cold-blooded" life forms such as lizards or fish, whose body temperatures are dictated by the external environment. For mammals and other homeothermic organisms, changes in enzyme reaction rates with temperature assume physiological significance only under conditions such as fever or hypothermia.
ASSAYS OF ENZYME-CATALYZED REACTIONS TYPICALLY MEASURE THE INITIAL VELOCITY
SUBSTRATE CONCENTRATION AFFECTS REACTION RATE
Only in the cross-hatched region do both the enzyme and the substrate carry an appropriate charge. Representation of an enzyme in the presence of a concentration of substrate that is below K m (A), at a.
THE MICHAELIS-MENTEN & HILL EQUATIONS MODEL THE EFFECTS OF SUBSTRATE CONCENTRATION
The Michaelis-Menten Equation
Double reciprocal or Lineweaver-Burke plot of 1/vi versus 1/[S] used to estimate Km and Vmax. The greatest virtue of the Lineweaver-Burk plot is that it can be used to determine the kinetic mechanisms of enzyme inhibitors (see below).
The Catalytic Constant, K cat
However, in using a double reciprocal plot to determine kinetic constants, it is important to avoid introducing bias through binning the data at low values of 1/[S]. Alternatively, a single reciprocal plot such as Eadie-Hofstee (vi vs. vi/[S]) or Hanes-Woolf ([S]/vi vs. [S]) can be used to minimize clustering.
Catalytic Efficiency, k cat /K m
The Hill Equation Describes the Behavior of Enzymes that Exhibit Cooperative Binding of Substrate
Binding of substrate to one site then increases the affinity of the remaining sites to bind additional substrate. A graphical representation of a linear form of the Hill equation is used to evaluate S50, the substrate concentration that produces half-maximal velocity, and the degree of cooperativity n.
KINETIC ANALYSIS DISTINGUISHES COMPETITIVE FROM NONCOMPETITIVE INHIBITION
Competitive Inhibitors Typically Resemble Substrates
A competitive inhibitor and a substrate exert reciprocal effects on the concentration of EI and ES complexes. Since the formation of ES complexes removes free enzyme available to combine with the inhibitor, increasing [S] decreases the concentration of the EI complex and increases the reaction rate.
Double-Reciprocal Plots Facilitate the Evaluation of Inhibitors
For simple noncompetitive inhibition, E and EI possess identical affinity for substrate, and the EIS complex generates product at a negligible rate (Figure 8–11). More complex noncompetitive inhibition occurs when binding of the inhibitor affects the apparent affinity of the enzyme for substrate, causing the lines to cross in the third or fourth quadrant of a double-reciprocal graph (not shown).
Dixon Plot
While some inhibitors exhibit characteristics of a mixture of competitive and noncompetitive inhibition, evaluation of these inhibitors is beyond the scope of this chapter. Unlike the equilibrium dissociation constant Ki, the numerical value of IC5 0 varies as a function of the specific circumstances of substrate concentration, etc., under which it is determined.
Tightly Bound Inhibitors
Irreversible Inhibitors "Poison" Enzymes
Mechanism-Based Inhibition
Neither the Lineweaver-Burk nor the Dixon approach is applicable, as suicide inhibitors violate a key boundary condition common to both approaches, namely that the activity of the enzyme does not decrease over the course of the assay.
MOST ENZYME-CATALYZED REACTIONS INVOLVE TWO OR MORE SUBSTRATES
Sequential or Single-Displacement Reactions
Ping-Pong Reactions
Most Bi-Bi Reactions Conform to Michaelis-Menten Kinetics
Product inhibition studies are used to complement kinetic analyzes and to distinguish between ordered and random Bi-Bi reactions. For example, in a random order Bi-Bi reaction, each product will be a competitive inhibitor regardless of which substrate is designated as the variable substrate.
KNOWLEDGE OF ENZYME KINETICS, MECHANISM, AND INHIBITION AIDS DRUG DEVELOPMENT
However, for a sequential mechanism (Figure 8–13, top), only product Q will produce the pattern indicative of competitive inhibition when A is the variable substrate, while only product P will produce this pattern with B as the variable substrate. The other combinations of product inhibitor and variable substrate will produce forms of complex non-competitive inhibition.
Many Drugs Act as Enzyme Inhibitors
An increase in the concentration of one substrate (S1), while holding the concentration of the other substrate (S2) constant, changes both the x- and y-intercepts, but not the slope. For example, statin drugs lower cholesterol production by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase (Chapter 26), while emtricitabine and tenofovir disoproxil fumarate block human replication.
Enzyme Kinetics Defines Appropriate Screening Conditions
Stop or inhibit aberrant molecular processes caused by genetic, environmental or biological stimuli with minimal disruption of the host's normal cellular functions. Based on their diverse physiological roles and high degree of substrate selectivity, enzymes constitute natural targets for the development of pharmacological agents that are both potent and specific.
Many Drugs Are Metabolized In Vivo
The effects of simple competitive inhibitors, which typically resemble substrates, are overcome by raising the concentration of the substrate. Knowledge of factors that control the speed of enzyme-catalyzed reactions is thus essential for an understanding of the molecular basis of the disease.
REGULATION OF METABOLITE FLOW CAN BE ACTIVE OR PASSIVE
Disruption of the sensor response mechanism responsible for maintaining homeostatic balance can be detrimental to human health. The differential response of the rate of an enzyme-catalyzed reaction, V, to the same step change in substrate concentration at substrate concentration Km (VA) or well above Km (VB).
Metabolite Flow Tends to Be Unidirectional
COMPARTMENTATION ENSURES METABOLIC EFFICIENCY &
SIMPLIFIES REGULATION
Controlling an Enzyme That Catalyzes a Rate-Limiting Reaction Regulates an Entire Metabolic Pathway
REGULATION OF ENZYME QUANTITY
Proteins Are Continually Synthesized and Degraded
While the steady-state concentrations of some enzymes and other proteins remain essentially constant, or. The absolute amount of an enzyme reflects the net balance between the rate of its synthesis and the rate of its degradation.
Control of Enzyme Synthesis
In humans, changes in the levels of specific enzymes can be affected by a change in the rate constant of the overall processes of synthesis (k2), degradation (kdeg), or both.
Control of Enzyme Degradation
Recognition by proteolytic enzymes can also be regulated by covalent modifications such as phosphorylation; binding of substrates or allosteric effectors; or binding to membranes, oligonucleotides or other proteins.
MULTIPLE OPTIONS ARE AVAILABLE FOR REGULATING CATALYTIC ACTIVITY
ALLOSTERIC EFFECTORS REGULATE CERTAIN ENZYMES
Curved red arrows represent feedback loops and indicate sites of feedback inhibition of specific end products. The effect of an excess of two or more end products may be strictly additive or, alternatively, greater than their individual effect (cooperative feedback inhibition).
Aspartate Transcarbamoylase Is a Model Allosteric Enzyme
Thus, for this pathway, excess B limits synthesis of all four end products, regardless of the need for the other three. Superimposed on simple feedback loops (dashed red arrows) are several feedback loops (solid red arrows) that regulate enzymes common to biosynthesis of various end products.
Allosteric & Catalytic Sites Are Spatially Distinct
FEEDBACK REGULATION IS NOT SYNONYMOUS WITH FEEDBACK INHIBITION
MANY HORMONES ACT THROUGH ALLOSTERIC SECOND MESSENGERS
REGULATORY COVALENT MODIFICATIONS CAN BE REVERSIBLE OR IRREVERSIBLE
PROTEASES MAY BE SECRETED AS CATALYTICALLY INACTIVE PROENZYMES
Selective proteolysis changes a proprotein by one or more successive proteolytic "cuts" to a form that exhibits the characteristic activity of the mature protein, for example its enzymatic activity. Proteins synthesized as proproteins include the hormone insulin (proprotein = proinsulin), the digestive enzymes pepsin, trypsin and chymotrypsin (proproteins = .pepsinogen, trypsinogen and chymotrypsin respectively), various factors of the blood coagulation and blood clot dissolution cascades (see Chapter 51), and the connective tissue protein collagen (proprotein = procollagen).
Proenzymes Facilitate Rapid Mobilization of an Activity in Response to Physiologic Demand
Activation of Prochymotrypsin Requires Selective Proteolysis
Two-dimensional representation of the sequence of proteolytic events that ultimately result in the formation of the chymotrypsin catalytic site, which includes the catalytic triad Asp 102-His57-Ser195 (see Figure 7–7).
REVERSIBLE COVALENT MODIFICATION REGULATES KEY MAMMALIAN ENZYMES
Phosphorylation-dephosphorylation allows the functional properties of the affected enzyme to be changed only as long as it fulfills a specific need. A second factor underlying the widespread use of protein phosphorylation-dephosphorylation lies in the chemical properties of the phosphoryl group itself.
Covalent Modification Regulates Metabolite Flow
The ease of interconversion of enzymes between their phospho- and dephospho-forms accounts in part for the frequency with which phosphorylation-dephosphorylation is used as a mechanism of regulatory control. To change the functional properties of an enzyme, any change to its chemical structure must affect.
PROTEIN PHOSPHORYLATION IS EXTREMELY VERSATILE
Phosphorylation-dephosphorylation at any site can be catalyzed by multiple protein kinases or protein phosphatases. Many protein kinases and most protein phosphatases act on more than one protein and are themselves converted between active and inactive forms by binding of second messengers or by covalent modification by phosphorylation-dephosphorylation.
INDIVIDUAL REGULATORY EVENTS COMBINE TO FORM SOPHISTICATED CONTROL NETWORKS
This state or degree of phosphorylation is determined by the relative activities of the protein kinase and protein phosphatase, a reflection of the presence and relative strength of the environmental signals acting through each. This facilitates passive control of the rate of product formation in response to changes in levels of metabolic intermediates.
GENOMICS: AN INFORMATION AVALANCHE
Rather than having one or more specific causative agents, the presence of which is both necessary and sufficient, the appearance and development of the aforementioned diseases reflects the complex interplay between the individual's genetic makeup, diet and lifestyle, as well as a number of environmental factors such as the presence of toxins, viruses or. The sheer amount of information that needs to be processed to understand, as completely and comprehensively as possible, the molecular mechanisms that underlie the behavior of living organisms, as well as the way in which perturbations can lead to disease or dysfunction, is far beyond the ability. of the human mind to review and analyze.
The Human Genome Project
High-performance sequencers, powerful computer programs and an element of competition contributed to the rapid progress of both groups, culminating in a fast finish to the HGP.
Genomes and Medicine
The opportunities offered by the advancing genomic revolution will present society with profound challenges in the areas of ethics, law, and public policy. The first harbingers of these challenges can be seen in the ongoing controversies surrounding genetically modified foods, the cloning of whole animals, and the use of human embryonic stem cells in research.
BIOINFORMATICS
BIOINFORMATIC AND GENOMIC RESOURCES
Genbank, Uniprot, and the Protein Database
The Hapmap Database
In 2002, researchers from the United States, Canada, China, Japan, Nigeria, and the United Kingdom launched the International HapMap Project (http://www.hapmap.org), a comprehensive effort to identify SNPs. These genetic markers will also provide labels to identify and track specific genes as researchers seek to learn more about the critical processes of genetic inheritance and selection.
The ENCODE Project
Entrez Gene and dbGAP
COMPUTATIONAL BIOLOGY
IDENTIFICATION OF PROTEINS BY HOMOLOGY
Multiple sequence alignment algorithms analogously identify conserved nucleotide and amino acid letters in DNA, RNA, and polypeptides. For example, blastp compares an amino acid query sequence to a protein sequence database, blastn compares a nucleotide query sequence to a nucleotide sequence database, blastx compares a nucleotide query sequence translated in all reading frames to a protein sequence database to reveal potential translation products, tblastn compares a protein query sequence to a nucleotide sequence database dynamically translated into all six reading frames, and tblastx compares the six-frame translations of a nucleotide query sequence with the six-frame translations of a nucleotide sequence database.
IDENTIFICATION OF "UNKNOWN" PROTEINS
The space-filling representation of the enzyme HMG-CoA reductase and its complex with the drug lovastatin, shown in Figure 10-2, provides some perspective on the challenges inherent in identifying ligand binding sites from scratch. Shown are three-dimensional representations of the homodimeric HMG-CoA reductase from Pseudomonas mevalonii with (right) and without (left) the statin drug lovastatin bound.
COMPUTER-AIDED DRUG DESIGN
Three of the many different conformations of glucose, commonly referred to as chair (top), boat-turned-boat (middle), and half-chair (bottom), are shown. If no structural model is available for the protein of interest, computers can be used to aid the search for high-affinity inhibitors by calculating and designing structure-activity relationships (SARs).
SYSTEMS BIOLOGY & VIRTUAL CELLS
CONCLUSION
Ekins S, Mestres J, Testa B: In silico pharmacology for drug discovery: methods for virtual ligand screening and profiling. Hotopp JCD et al: Comparative genomics of Neisseria meningitides: nuclear genome, islands of horizontal transmission and pathogen-specific genes.
FREE ENERGY IS THE USEFUL ENERGY IN A SYSTEM
Biologic Systems Conform to the General Laws of Thermodynamics
If G is negative, the reaction proceeds spontaneously with loss of free energy; i.e. it is exergonic. When the reactants are present at concentrations of 1.0 mol/L, G0 is the standard free energy change.
ENDERGONIC PROCESSES PROCEED BY COUPLING TO EXERGONIC PROCESSES
On the other hand, if G is positive, the reaction proceeds only if free energy can be obtained; i.e. it is endergonic. Transfer of free energy from an exergonic to an endergonic reaction via a high-energy intermediate.
HIGH-ENERGY PHOSPHATES PLAY A CENTRAL ROLE IN ENERGY CAPTURE AND TRANSFER
The intermediate value for the free energy of hydrolysis of ATP has an important bioenergetic significance.
The Intermediate Value for the Free Energy of Hydrolysis of ATP Has Important Bioenergetic Significance
High-Energy Phosphates Are Designated by