Proteins
4.4 Proteins as enzymes
+ ENZYME
ENZYME
SUBSTRATE
SUBSTRATE
+
(a)
(b)
Figure 4.8 Enzyme and substrate binding to form the enzyme substrate complex. In example (a), the enzyme and substrate fit similar as to a how a key fits in a lock.
In example (b), both the enzyme and substrate undergo a conformational change when they come into contact so that binding can occur
binding site and specific residues which act as the catalytic site.
The term active site is often used to refer col- lectively to both the binding and catalytic domains of the protein. Such sites have a particular shape and charge, which allows the enzyme to bind to highly specific substrates. Enzymes have been sug- gested to bind to their specific substrates similar to how a key fits in a lock. Alternatively, both the enzyme and substrate undergo a conformational change when their surfaces touch, such that they now fit each other accurately (see Figure 4.8).
4.4.2 Factors affecting rates of enzymatic reactions
The ability of the cell to increase or decrease the rate of enzymatic reactions is particularly important, so that the cell can tightly regulate the flow of biomolecules through energy-producing or energy-consuming pathways, according to the demands placed upon it (e.g. exercise). There are a number of important factors which can all affect the rate of an enzymatic reaction, and these are described below.
Substrate concentration
Increasing the substrate concentration while keep- ing enzyme concentration constant will acceler- ate the reaction rate, as more substrate molecules are now available to bind to the enzyme within a given time. This relationship is shown graphi- cally in Figure 2.9. However, it is important to note that the relationship is only linear over the initial period of the reaction, after which point the curve becomes hyperbolic. Eventually, there comes a point at which increasing substrate con- centration any further does not cause any further increase in reaction rate. At this substrate concen- tration, the enzyme is said to have saturated and has reached itsmaximal velocity (VMAX). At such points, the enzyme is working as fast as it can in converting the substrate to the product.
Also shown in Figure 4.9 is the Michaelis constant (KM), defined as the substrate concen- tration required to achieve half of the maximal velocity of an enzymatic concentration. The smaller the KM value, the greater affinity the enzyme has for its substrate and, as such, the enzyme is active even when the substrate concen- tration is low. Within our cells, most substrates are generally present at concentrations equal to or less than the KMvalue. This is beneficial, as it means that the enzyme can still respond to subtle
KM
Substrate Concentration
Reaction Rate
Vmax Vmax
1 2
Figure 4.9 Effect of substrate concentration on reac- tion rate
4 6 8 pH
Reaction Rate
10 12
Figure 4.10 Effect of pH on reaction rate
changes in substrate concentration, since it is still on the steep part of the curve.
pH
Cellular changes in pH can alter the affinity of an enzyme for its substrate, as the change in ioniza- tion state (i.e. addition or removal of protons) of the enzyme can alter the structure and charge of the binding site of the enzyme protein. Changes in pH can also alter the substrate directly, thus in turn influencing the rate of the reaction.
Enzymes usually function optimally over a nar- row range of pH that is close to the physiological pH of the particular cell (see Figure 4.10). For example, enzymes in the stomach usually func- tion optimally close to a pH of 2, given the acidic conditions present. In contrast, the enzymes in our muscles usually function optimally around 7.
While cellular changes in pH are usually small, skeletal muscle shows the largest change in pH, especially during high-intensity exercise condi- tions, where pH can fall from 7.1 to 6.6 (Bogdanis et al., 1995). For this reason, an acidosis-induced inhibition of metabolic enzymes involved in the glycolytic pathway is often associated with fatigue during high-intensity exercise (Hargreaves et al., 1998).
Temperature
One of the most profound factors influencing rates of enzymatic reactions is cell temperature, as this increases the kinetic energy of the reactants, thus
0 20
Temperature ( C)
Reaction Rate
40 60
Figure 4.11 Effect of temperature on reaction rate
raising the chances of effective collisions. Rates of reaction show a linear increase up until approxi- mately 50◦C, after which point the enzyme protein denatures (i.e. loses its three-dimensional struc- ture) and loses function (see Figure 4.11).
Such sensitivity to temperature underpins our need to actively warm-up prior to exercise, so as to increase muscle temperature and increase enzyme activity in our muscles. Indeed, muscle tempera- ture can rise from around 35◦C at rest to 41◦C dur- ing intense exercise (Morton et al., 2006). To put this into a sporting performance context, profes- sional soccer players typically cover less distance in the first five-minute period of the second half period, compared with the last five min of the first half, and this has been suggested to be due to a fall in muscle temperature to near resting values during the half-time period (Mohr et al., 2004). In such instances, the same researchers also observed that performing light exercise during half-time to keep muscle temperature (and enzymes active) high can offset such performance decrements.
Enzyme concentration
Once the enzyme is saturated with substrate and working at VMAX, increasing the enzyme concen- tration itself will further augment reaction rate, as there are now more active sites available for sub- strate binding. Although increasing enzyme con- centration will increase VMAX, it is important to note that there is no concomitant increase in KM. (see Figure 4.12).
n io t a tr en c on ec ym nz de se ea cr In
on i t ra t en c on ec ym nz le ma or N
KM
Vmax
Substrate Concentration
Reaction Rate
Vmax
Figure 4.12 Effect of enzyme concentration on reac- tion rate
The ability of cells to adjust enzyme con- centration in order to increase reaction rate is one of the underpinning mechanisms by which skeletal muscle adapts to endurance training. With repeated bouts of endurance training, our muscle cells respond by making new proteins through the process of protein synthesis (see later sections), thus increasing enzyme concentration so that metabolic reactions operate at a greater rate. As such, the stress of exercise is reduced for a given absolute intensity (Holloszy & Coyle, 1984).
4.4.3 Coenzymes and cofactors
Many enzymes require the presence of additional reactive groups known as cofactors in order to have full catalytic function. Cofactors may con- sist of inorganic molecules such as the metal ions of zinc, copper, manganese, magnesium, etc. In such instances, these cofactors directly alter the binding activity of the enzyme by changing the charge distribution and shape of the active site of the enzyme. Where cofactors are tightly bound to the enzyme at all times, they are referred to as prosthetic groups, e.g. copper, manganese, zinc, biotin, vitamin B6, etc.
Alternatively, cofactors may be organic molecules known as coenzymes. In contrast to inorganic cofactors, coenzymes do not alter the enzyme’s binding activity but instead act as important compounds which directly participate in the reaction. Coenzymes can therefore be
considered to act as second substrates for or products of the reaction. For example, in the reaction outlined in Figure 4.6, where lactate is oxidized to pyruvate via the enzyme LDH, nicotinamide adenine dinucleotide (NAD+) acts as a coenzyme necessary for the reaction to proceed.
Many of the water-soluble vitamins, especially the B vitamins, are precursors (i.e. form the basic components) for coenzymes, and thus it is essential that we obtain the appropriate amount of vitamin B in our diets. Indeed, there are many diseases associated with vitamin B deficiency, given that certain enzymes cannot function optimally if they lack the necessary coenzyme. Members of the vita- min B family and the coenzymes they form are shown in Table 4.2, as are recommended dietary allowances (RDA) for 19–30 year old males (as recommended by the Food and Nutrition Board of the National Academy of Sciences). The coen- zymes NAD+ and FAD are especially important foroxidation-reduction reactions, as they form the basis for oxidative phosphorylation, the energy- producing pathway which is dominant during pro- longed endurance-type exercise.
4.4.4 Classification of enzymes
As we saw in Chapter 3, there are many common types of chemical reaction. Similarly, the enzymes which facilitate many of these reactions can also be classified into six common classes and sub-classes (see Table 4.3), as designated by the International Union of Biochemistry. Most enzymes are recognizable by the suffix-ase, while the first part of the enzyme’s name (everything that precedes the suffix) usually refers to the type of reaction and/or the substrate which they act upon. For example, the enzyme creatine kinase has phosphocreatine (PCr) as its substrate and, as a kinase, it transfers a phosphate group to ADP, thus making the products ATP and creatine (Cr). This reaction is highly active within the first 10 seconds of maximal exercise, providing a rapid source of ATP production to fuel muscle contraction (Parolinet al. 1999).
Table 4.2 The B vitamin family and the coenzymes they form
B vitamin Coenzyme Abbreviation RDA
Thiamine (B1) Thiamine pyrophosphate TPP 1.2 mg/d
Riboflavin (B2) Flavin adenine dinucleotide FAD 1.3 mg/d Niacin (B3) Nicotinamide adenine dinucleotide NAD+ 16 mg/da
Vitamin B6 Pyridoxal phosphate PLP 1.3 mg/d
Pantothenic acid Coenzyme A CoA 5 mg/d
Folate (folacin) Tetrahydrofolic acid THFA 400µg/db
Biotin Biotin n/a 30µg/d
Vitamin B12 Methyl cobalamin n/a 2.4µg/d
avalues expressed as Niacin equivalents;
bvalues expressed as dietary folate equivalents; d (day)
Table 4.3 Enzyme classes and sub-classes and descriptions of their general functions Enzyme class Sub-class General function
Oxidoreductases Dehydrogenases Catalyze oxidation and reduction reactions Oxidases
Oxygenases Reductases Peroxidases Hydroxylases Transferases Kinases
Transcarboxylases Transaminases
Catalyze the transfer of elements from one molecule or compound to another
Hydrolases Phosphatases Esterases Peptidases
Catalyze reactions where cleavage of bonds is achieved by adding water
Lyases Synthases
Deaminases Decarboxylases
Catalyze reactions in which groups of elements are removed to form a double bond or are added to an existing double bond
Isomerases Mutases Isomerases Epimerases
Catalyze reactions that result in rearrangement of the structure of molecules
Ligases Synthetases Carboxylases
Catalyze bond formation between two substrate molecules
4.4.5 Regulation of enzyme activity
We have seen thus far how the rates of enzy- matic reaction can be altered by changing the cell temperature and pH as well as the substrate con- centration and enzyme concentration itself. The provision of additional reactive groups in the form of cofactors and coenzymes can also modify reac- tion rates. However, there are two other major cellular mechanisms by which enzyme activity can be further modified.
Covalent modification
The most rapid way to modify enzyme activ- ity is through the addition or removal of a phosphate group (which is provided from ATP) to the hydroxyl part of the amino acid side chains of serine, threonine or tyrosine residues (see Figure 4.13). Such processes alter the conformation of the enzyme and are known as phosphorylation (addition of phosphate) or dephosphorylation (removal of phosphate). This form of covalent modification of enzymes is in turn regulated by enzymes known as protein kinases (responsible for phosphorylation) or protein phosphatases(responsible for dephosphorylation).
Phosphorylation-dephosphorylation has been likened to turning on and off a light switch, as it rapidly alters enzyme activity and it also has an
‘all or none’ effect – the enzyme is either active or inactive (Houston, 2006). Usually the enzyme is active when in the phosphorylated state, though there are many examples when enzymes are
P O−
O− O O Protein Kinase
Protein Phosphatase
ADP
Phosphorylated Dephosphorylated
H2O Pi
Enzyme Enzyme
ATP
Figure 4.13 Regulation of enzyme activity through phosphorylation and dephosphorylation
actually active in the dephosphorylated state. For example, glycogen synthase, the enzyme respon- sible for glycogen synthesis, is inactive when phosphorylated and requires dephosphorylation by protein phosphatase 1 to become active.
The overall mechanism of controlling enzyme activity by phosphorylation or dephosphorylation is critical to sport and exercise metabolism, and there will be many examples of this type of regu- lation in subsequent chapters.
Allosteric modification
Whereas phosphorylation-dephosphorylation gen- erally renders the enzyme as active or inactive, enzymes can also gradually grade their activity via allosteric regulation. In this process, small molecules known as allosteric effectors bind to regulatory domains other than the active site.
This, in turn, alters the shape and/or charge of the active site (see Figure 4.14). In this way, the enzyme can therefore increase or decrease its affinity for binding of substrates, depending on whether positive or negative allosteric effectors have bound, respectively. If phosphorylation- dephosphorylation can be likened to turning on and off a light switch, allosteric regulation can be thought of as a dimmer switch, where the enzyme’s activity can be fine tuned along a continuum of activity (Houston, 2006).
ALLOSTERIC
ACTIVATOR ALLOSTERIC
ACTIVATOR ACTIVE ENZYME INACTIVE
ENZYME
SUBSTRATE SUBSTRATE
Figure 4.14 Regulation of enzyme activity through allosteric modification. Binding of the allosteric effector to the non-binding site alters the shape and/or charge of the binding site, thus increasing the enzyme’s affinity for binding to its substrate
Similar to covalent regulation, allosteric regula- tion of enzymes is highly important for sport and exercise metabolism, in order to regulate the rate of energy provision according to the intensity and duration of the exercise. In such circumstances, products of energy-producing reactions such as ADP, AMP, Pi and H+, etc. can all act as a feedback loop mechanism to fine tune the rate of enzymatic reactions during exercise (Parolin et al., 1999). This will be highlighted in much more detail in future chapters, where we will examine the regulation of metabolism during different types of exercise.