Search and Analysis of Enzyme Data

TABLE 7.4. pH Effects on Enzyme Kinetics

Diagnostic pH-Rate

Profile Rate Expression Low pH High pH

Full bell K:KK/(H/K;1;K/H) logK:logKK9pK;pH logK:logKK;pK9pH Left half bell K:KK/(H/K;1) log K:log KK9pK;pH

Right half bell K:KK/(1;K/H) log K:log KK;pK9pH

Full bell V:VK/(H/K;1;K/H) log V:logVK9pK;pH log V:log VK;pK9pH Left half bell V:VK/(H/K;1) log V:log VK9pK;pH

Right half bell V:VK/(1;K/H) log V:log VK;pK9pH

Note: K and K or K and K are ionizing group(s) in the free enzyme or the enzyme—substrate complex, respectively.

3. Change in the ionization of substrate(s)

4. Change in the ionization of other groups in the enzyme 5. Denaturation of the enzyme

The pH effect on kinetic parameters (pH-rate/binding profile) may provide useful information on the ionizaing groups of the enzyme if the kinetic studies are carried out with nonionizable substrate in the pH region(pH 5—9) where enzyme denaturation is minimum. If the Michaelis constant (K) and/or the maximum velocity(V ) vary with pH, the number and pK values of the ionizing group(s) can be inferred from the shape of pH-rate profile(pH versus pK and pH versus log V plots), namely, full bell shape for two ionizing groups and half bell shape for one ionizing group(see Table 7.4).

The initial rate enzyme kinetics uses very low enzyme concentrations (e.g., 0.1M to 0.1 pM) to investigate the steady-state region of enzyme-catalyzed reac-tions. To investigate an enzymatic reaction before the steady state (i.e., transient state), special techniques known as transient kinetics (Eigen and Hammes, 1963) are employed. The student should consult chapters of kinetic texts (Hammes, 1982;

Robert, 1977) on the topics. KinTekSim (http://www.kintek-corp.com/kintek-sim.htm) is the Windows version of KINSIM/FITSIM (Frieden, 1993) which analyzes and simulate enzyme-catalyzed reactions.

7.3. SEARCH AND ANALYSIS OF ENZYME DATA

Figure 7.3. Enzyme nomenclature database of ExPASy.

·

Enzyme for the information on enzyme molecules and enzymatic reactions

·

Compound for the information on metabolites

·

Reaction for the collection of substrate—product relationships

Select Search enzymes and compounds under DBGet/LinkDB Search to open query page(Figure 7.4). Enter the enzyme name or the substrate name in the bfind mode and click the Submit button. From the list of hits, select the desired entry by clicking the ECname. This returns information on name, class, reaction, pointers to structures of substrates/products/cofactor, links to pathway for which the selected enzyme is the member enzyme of the pathway, and related databases.

BRENDA (Schomburg et al., 2002) is the comprehensive enzyme information system that can be accessed at http://www.brenda.unikoeln.de/. Select New Query Forms to initiate Search by ECnumber, by Enzyme name or by Organism(Figure 7.5). Enter the ECnumber or the enzyme name (you can use * for partial name, e.g.,

*kinase) and click Query. From the list of hits (only one entry is returned with EC number), select the desired entry by clicking the ECnumber. The request returns the following information (the available information differs with each enzyme, e.g., for lysozyme): ECnumber, Organism, Systematic name, Recommended name, Synony-ms, CAS registry number, Reaction, Reaction type, Substrates/products, Natural substrate, Turnover number [1/min], Specific activity [mol/min/mg], KK value [mM], pH optimum, pH range, Temperature optimum [°C], Temperature range [°C], Cofactors, Prosthetic groups, Activating substances, Metal/ions, Inhibitors, Source/tissue, Localization, Purification, Crystallization, Molecular weight, Subunits,

134 DYNAMIC BIOCHEMISTRY: ENZYME KINETICS

Figure 7.4. LIGAND database. A composite database for searching/retrieving enzyme information by enzyme name, EC number, substrates/products, and reactions.

Figure 7.5. Search enzyme information at Brenda. Brenda is the comprehensive enzyme database for retrieving chemical, kinetic, and structural properties of enzymes via EC number, enzyme name, and organism (biological source). The search page by EC number is shown.

SEARCH AND ANALYSIS OF ENZYME DATA 135

Figure 7.6. Enzymology database, EMP. The enzyme data published in literatures can be searched/retrieved from EMP via enzyme name/EC code and biological sources.

Cloned, pH stability, Temperature stability [°C], Organic solvent stability, Oxida-tion stability, General stability, Storage stability, Renatured, and Links to other databases and references.

EMP at http://wit.mcs.anl.gov/EMP/ is the resource site for summarized enzyme data that have been published in the literature. The site opens with the Simple query form (Figure 7.6). Enter the enzyme name into the name query box of ‘Find an enzyme,’ select ‘Common name,’ then enter the common organism name for ‘In an organism or taxon,’ and enter tissue name in response to ‘Extracted from.’ Clicking Submit Query returns an itemized summary of published enzyme data (data from one article may appear in more than one entries for different substrates) including concise assay and purification procedures, kinetic equations and kinetic parameters.

The Enzyme Structure Database (http://www.biochem.ucl.ac.uk/bsm/enzymes/

index.html), which contains the known enzyme structures of PDB, can be searched via ECnumber hierarchically(Figure 7.7). The search returns a list of individual pdb files with a link to CATH and pointers to PDBsum, ExPaSy, KEGG, and WIT.

Clicking PDBsum opens the PDBsum page, which contains descriptive headings of enzyme, CATH classification, amino acid sequence with secondary structure desig-nation, clickable PROMOTIF summary, TOPS(protein topology cartoon of related representative enzyme), PROSITE patterns, MolScript picture, as well as graphical presentations of ligand/ligand—active-site interactions. Click LIGPLOT of interac-tions(under Ligand) to display ligand—active site interactions (Figure 7.8). Pressing the RasMol button changes the view window into the RasMol window(if RasMol

136 DYNAMIC BIOCHEMISTRY: ENZYME KINETICS

Figure 7.7. Home page of enzyme structure database. Links to enzyme structure and analysis servers are available at the Enzyme Structure Database which extracts and collects enzyme structures from pdb files.

Figure 7.8. The substrate interaction at the active site of an enzyme. The interaction of tetra-N,N,N,N-acetylchitotetraose (NAG₄) with amino acid residues at the active site of lysozyme (1LZC.pdb) can be viewed/saved at PDBsum server (Enyme Structure Database;PDBsum;LIGPLOT of interactions under Ligand) linked to the Enzyme Struc-ture Database.

Figure 7.9. Starting page of Leonora.

is installed). Right click on the window to open the menu box (File, Edit, Display, Color, Options, and Rotation). The coordinate file of the ligand—active-site interaction can be saved(PDB format or MDL mol format) by selecting File;Save Molecule As. The PDBsum can be accessed directly at http://

www.biochem.ucl.ac.uk/bsm/pdbsum/.

7.3.2. Analysis of Kinetic Data

For the statistical and computer analysis of enzyme kinetic data, the students should consult published articles on the topics (Cleland, 1967; Crabble, 1992; Wilkinson, 1961). The software DynaFit, applicable to enzyme kinetic analysis, has been described(Chapter 6). In this chapter the program Leonora, which accompanies the text Analysis of Enzyme Kinetic Data by A. Cornish-Bowden (Cornish-Bowden, 1995), will be used to perform regression analysis of enzyme kinetic data. The software can be downloaded from http://ir2lcb.cnrs-mrs.fr/¨athel/leonora0.htm. After installation, launch the program(MS-DOS) to open the Main menu providing a list executable commands (Figure 7.9). Type D (select Data) to bring the Data menu, and type I(select Input new data) to enter kinetic data. Use Tab key to move across the columns and arrow keys to move up and down the rows. Enter label on the row 1 and data for the others. Press Esc to complete the data entry. Furnish short description for the Title, type N to enter filename, and save the data file (.mmd).

Type X to exit the Data menu and return to the Main menu. Type Q (Equation) to select the appropriate rate equation from the pop-up Model menu (the list differs depending on kinetic data file). The menu entries of the equations are listed in Table 7.5.

To save the kinetic results, key in O for Output requirement and then R for Result page(Figure 7.10), which lists fitted kinetic parameters. Exit to the Calcula-tions menu by typing C. Define method and weighting system, then C to calculate

138 DYNAMIC BIOCHEMISTRY: ENZYME KINETICS

Figure 7.10. Result page of Leonora.

TABLE 7.5. Representative Menu Entries of Kinetic Equations in Leonora

Equations by Name Algebraical Equations

Michaelis—Menten M: v: V [S]/(KK;[S])

Substrate inhibition S: v: V [S]/(KK;[S](1;[S]/KQG)) Michaelis-Menten(ignoring [I]) M: v: V [S]/(KK;[S])

Primary Michaelis— Menten(at each [I]) P: v: V [S]/(KK ;[S]) Generic inhibition,(at each [S]) G: v: v/(1 ; [I]/KG)

Competitive inhibition C: v: V [S]/(KK(1;[I]/KGA);[S]) Uncompetitive inhibition U: v: V [S]/(KK;[S](1;[I]/KGS))

Mixed inhibition I: v: V [S]/(KK(1;[I]/KGA);[S](1;[I]/KGS)) Michaelis—Menten(ignoring [B]) M: v: V [A]/(KK;[A])

Michaelis—Menten(ignoring [A]) I: v: V [B]/(KK;[B]) Primary Michaelis— Menten(at each [B]) P: v: V [A]/(KK ;[A]) Primary Michaelis— Menten(at each [A]) R: v: V [A]/(KK ;[A])

Substituted enzyme mechanism S: v: V [A][B]/(KK [A];KK[B];[A][B]) Ternary-complex mechanism T: v: V [A][B]/(K ;KK [A];KK[B];[A][B]) Ordered equilibrium mechanism O: v: V [A][B]/(K ;KK [A];[A][B])

S-shaped pH profile S: k: K /(1;[H>]/K)

Z-shaped pH profile Z: k: K /(1;K/[H>])

Bell-shaped pH profile B: k: K /(1;[H>]/K;K/[H>])

Notes: Reprinted from table 9.1(p. 156) from Analysis of Enzyme Kinetic Data by Athel Cornish-Bowden (1995) by permission of Oxford University Press.

1. The default Michaelis—Menten refers to uni uni or uni bi rate equation.

2. Mixed inhibition and noncompetitive inhibition can be used interchangeably.

3. Substituted-enzyme mechanism refers to ping pong bi bi mechanism.

4. Ternary-complex mechanism refers to order bi bi mechanism.

5. Michaelis—Menten, ignoring [X] refers to kinetic treatment of the bi bi reaction, A;B by ignoring the X (either A or B).

6. S-Shaped, Z-shaped and bell-shaped pH profiles refer to right-half, left-half and full bell profiles respectively.

best fit. To view the graphical results, type P for Plot results(which becomes active after calculations). Define Axes and Scale ranges. Use Tab key to move between abscissa and ordinate, and use arrow keys to define plotting parameters (e.g., 1/v).

Type P to Plot.

SEARCH AND ANALYSIS OF ENZYME DATA 139

Oxidoreductase Source Substrate Product Coenzy Stereo

Alcohol DH Horse liver Ethanol Acetaldehyde NAD A

Alcohol DH Yeast Ethanol Acetaldehyde NAD A

Alcohol DH(Aldehyde, Fruit fly Glycerol -Glyceraldehyde NADP A

RD)

Alcohol DH(Aldehyde, Human liver Glycerol -Glyceraldehyde NADP A

RD)

Homoserine DH Pea -Homoserine -Adpsemiald NADP B

Glycerol DH Aerobacter aerogenes Glycerol DiOHacetone NAD A

Glycerol DH Rabbit muscle Glycerol -Glyceraldehyde NADP A

Glycerol-3P DH E. coli SnGlycerol-3P DiOHacetone P NADP B

Glycerol-3P DH Rabbit muscle SnGlycerol-3P DiOHacetone P NAD B

XylitolDH(Xylu RD) Yeast Xylitol -Xylulose NAD B

Xylitol DH(Xylu RD) Pigeon liver Xylitol -Xylulose NADP B

Mannitol-1P DH E. coli -Mannitol-1P -Fructose-6P NAD B

Polyol DH(Aldose Human placenta -Sorbitol -Glucose NADP A

RD)

UDPGlucose DH Beef liver UDPGlucose UDPGlucuronate NAD B

Shikimate DH Pea Shikimate 5-DeHshikimate NADP A

-Lactate DH L actobacillus -Lactate Pyruvate NAD A

arabinosus

-Lactate DH L actobacillus -Lactate Pyruvate NAD A

arabinosus

Glycerate DH Spinach -Glycerate Hydroxyacetone NAD A

3-Hydroxybutyrate Beef heart -OHbutyrate Acetoacetate NAD B

DH

Malate DH Pig heart -Malate Oxaloacetate NAD A

Malate DH Pigeon liver -Malate Pyruvate NADP A

(decarboxyl)

Isocitrate DH Pea threo-Isocitrate -Ketoglutarate NAD A

Isocitrate DH Pea threo-Isocitrate -Ketoglutarate NADP A

P-Gluconate DH Yeast 6P--Gluconate -Ribulose-5P NADP B

Glucose DH Beef liver --Glucose Gluc--lactone NAD B

Galactose DH Pseud. fluorescens --Galactose Gal- -lactone NAD B

Glucose-6P DH L . mesenteroides -Glucose-6P Glu-lactone6P NAD B

Glucose-6P DH Yeast -Glucose-6P Glu-lactone6P NADP B

Aryl alcohol DH Rabbit kid cortex Benzyl alcohol Benzaldehyde NADP B

P-Glycerate DH E. coli P-OHPyruvate Glycerate-3P NAD A

Carnitine DH Pseud. aeruginosa Carnitine 3-DeHCarnitine NAD B