Prcticas de Bioqumica. Programa de Biologa.

Daro Mndez Cuadro, Ph D.

ENZYMES:
Kinetics and Structural Analysis
(Based in: Dynamic Biochemistry: Enzime Kinetics,
from An Introduction to Computational Biochemistry, by C. Stan Tsai)

Introduction
Enzymes are globular proteins whose sole function is to catalyze biochemical reactions. The most
important properties of all enzymes are their catalytic power, specificity, and capacity to
regulation.
1. CHARACTERISTICS OF ENZYMES
Some characteristics of enzymes can be summarized as follows:

All enzymes are considered proteins: The term enzyme refers to biological catalysts, which are
proteins with molecular weights generally ranging from 1.5x104 to 108 daltons. The nonprotein
biocatalysts such as catalytic RNA and DNA are known as ribozymes and deoxyribozymes
respectively, while engineered catalytic antibodies are called abzymes.
Enzymes increase the rate but do not influence the equilibrium of biochemical reactions:
Enzymes are highly efficient in their catalytic power displaying rate enhancement of 106 to 1012
times those of uncatalyzed reactions without changing the equilibrium constants of the
reactions.
Enzymes exhibit a high degree of specificity for their substrates and reactions: Enzymes are
highly specific both in the nature of the substrate(s) that they utilize and in the types of
reactions that they catalyze. Enzymes may show absolute specificity by a catalyzing reaction
with a single substrate. Enzymes may display chemical (bond) specificity by promoting
transformation of a particular chemical functional group (e.g., hydrolysis of esteric bond by
esterases or phosphorylation of primary hydroxy group of aldohexoses by hexokinase). The
stereospecificity refers to the ability of enzymes to choose only one of the enantiomeric pair
of a chiral substrate in chiral stereospecificity, such as D-lactate dehydrogenase versus L-
lactate dehydrogenase. One of the most subtle stereospecificities of enzymes relates their
ability to distinguish between two identical atoms/groups (proR versus proS) bonded to a
carbon atom in prochiral stereospecificity, such as proR glycerol- 3-phosphate dehydrogenase
versus proS glycerol-3-phosphate dehydrogenase.
Some enzymes require cofactors for the activities: Enzymes that require covalent cofactors
(prosthetic groups, e.g., heme in cytochromes) or noncovalent cofactors (coenzymes, e.g.,
NAD(P)+ in dehydrogenases) for activities are called haloenzymes (or simply enzymes). The
protein molecule of a haloenzyme is termed proenzyme. The prosthetic group/coenzyme
dictates the reaction type catalyzed by the enzyme, and the proenzyme determines the
substrate specificity.
The active site of an enzyme is the region that specifically interacts with the substrate: A
number of generalizations concerning the active site of an enzyme are as follows:
1. The active site of an enzyme is the region that binds the substrates (and cofactors, if any)
and contributes the catalytic residues that directly participate in the making and breaking of
bonds.
2. The active site takes up a relatively small part of the total dimension of an enzyme.
3. The active site is a three-dimensional entity and dynamic.
4. Active sites are generally clefts or crevices.
 Prcticas de Bioqumica. Programa de Biologa.
Daro Mndez Cuadro, Ph D.

5. Substrates are bound to the active site of enzymes by noncovalent bonds.

6. The specificity of binding depends on the precisely defined arrangement of atoms in an
active site.
7. The induced fit model of Koshland, (1958) has been proposed to explain how the active site
functions.
Enzymatic catalysis involves formation of an intermediate enzymesubstrate complex.
Enzymes lower the activation energies of reactions.
Enzymes are usually named with reference to the reaction they catalyze. It is customary to add the
suffix -ase to the name of its major substrate. The Enzyme Commission (EC) has recommended
nomenclature of enzymes based on the six major types of enzyme-catalyzed reactions
(http://www.chem.qmw.ac.uk/iubmb/enzyme/):

EC1: Oxidoreductases catalyze oxidationreduction reactions.

EC2: Transferases catalyze group transfer reactions.
EC3: Hydrolases catalyze hydrolytic reactions.
EC4: L yases catalyze cleavage and elimination reactions.
EC5: Isomerases catalyze isomerization reactions.
EC6: Ligases catalyze synthetic reactions.
Thus, the ECnumbers provide unique identifiers for enzyme functions and give us useful keyword
entries in database searches.
The ENZYME database at http://www.expasy.ch/enzyme/ provides information on ECnumber,
name, catalytic activity, and hyperlinks to sequence data of enzymes. The 3D structures of
enzymes can be accessed via Enzyme Structures Database at
http://www.biochem.ucl.ac.uk/bsm/enzyme/index.html. Some other enzyme databases are listed
in Table 1.

2. KINETICS OF ENZYMATIC REACTIONS

Enzyme kinetics investigates the rates of enzyme-catalyzed reactions as affected by various

factors, offers an enormous potential to the study of enzyme reaction mechanisms and functions.
Some important factors that affect the rates of enzymatic reactions are enzyme concentration,
ligand (substrates, products, inhibitors, and activators) concentrations, solvent (solution, ionic
strength, and pH), and temperature. When all these factors are properly analyzed, it is possible to
learn a great deal about the nature of enzymes.
The kinetic studies of an enzymatic reaction by varying ligand concentrations provide kinetic
parameters that are essential for an understanding of the kinetic mechanism of the biochemical
reaction. Operationally, the initial rate enzyme kinetics can be treated according to two
assumptions:

2.1 Quasi-equilibrium Assumption:

Quasi-equilibrium, also known as rapid equilibrium, assumes that an enzyme (E) reacts with
substrate (S) rapidly to form an enzymesubstrate complex (ES) (with a rate constant, k1) that
breaks down to release the enzyme and product (P). The enzyme, substrate, and the enzyme
 Prcticas de Bioqumica. Programa de Biologa.
Daro Mndez Cuadro, Ph D.

substrate complex are at equilibrium; that is the rate at which ES dissociates to E + S (rate constant
of k2 is much faster than the rate of forming E + P (rate constant of k3).

The general rule for writing the rate equation according to the quasi-equilibrium treatment of
enzyme kinetics was established by Michaelis and Menten for unimolecular reactions as show
above.

Table 1. Enzyme Databases

Web Site
ENZYME DB: General information
Enzyme structure database:
Structures
LIGAND: Enzyme reactions
Brenda: General enzyme data
EMP: General, literature
summary
Esther: Esterases
Merops: Peptidases
Protease
CAZy: Carbohydrate active
enzymes
REBASE: Restriction enzymes
Ribonuclease P database
PKR: Protein kinase
PlantsP: Plant protein kinases
and phosphatase
Aminoacyl-tRNA synthetases
MDB: Metalloenzymes
Promise: Prosthetic group/Metal
enzymes
Aldehyde dehydrogenase
G6P dehydrogenase
2-Oxoacid dehydrogenase
complex
URL
http://www.expasy.ch/enzyme/
http://www.biochem.ucl.ac.uk/bsm/enzyme/index.html
http://www.gebine.ad.jp/dbget/ligand.html
http://www.brenda.uni-koeln.de/
http://wit.mcs.anl.gov/EMP/
http://www.ensam.inra.fr/cholinesterase/
http://www.bi.bbsrc.ac.uk/Merops/Merops.htm
http://delphi.phys.univ-tours.fr/Prolysis
http://afmb.cnrs-mrs.fr/_pedro/CAZY/db.html
http://rebase.neh.com/rebase/rebase.html
http://www.mbio.ncsu.edu/RnaseP/home.html
http://pkr.sdsc.edu
http://PlantP.sdsc.edu
http://rose.man.poznan.pl/aars/index.html
http://metallo.scripps.edu/
http://bmbsgi11.leads.ac.uk/promise/
http://www.ucshc.edu/alcdbase/aldhcov.html
http://www.nal.usda.gov/fnic/foodcomp/
http://qcg.tran.wau.nl/local/pdhc.htm

2.2 Steady-State Assumption

The steady-state treatment of enzyme kinetics assumes that concentrations of the enzyme-
containing intermediates remain constant during the period over which an initial velocity of the
reaction is measured. Thus, the rates of changes in the concentrations of the enzyme-containing
species equal zero. Under the same experimental conditions (i.e., [S] [E] and the velocity is
measured during the very early stage of the reaction), the rate equation for one substrate reaction
(uni uni reaction), if expressed in kinetic parameters (V and Ks), has the form identical to the
MichaelisMenten equation.
 Prcticas de Bioqumica. Programa de Biologa.
Daro Mndez Cuadro, Ph D.

3. Environmental Effects

The rate of an enzymatic reaction is affected by a number of environmental factors, such as

solvent, ionic strength, temperature, pH, and presence of inhibitor/activator. Some of these
effects are described below.
3.1 Presence of Inhibitors: inhibition Kinetics:
The kinetic study of an enzymatic reaction in the presence of inhibitors is one of the most
important diagnostic procedures for enzymologists. The inhibition (reduction in the rate) of an
enzyme reaction is one of the major regulatory devices of living cells and offers great potentials for
the development of pharmaceuticals. An irreversible inhibitor forms the stable enzyme complex or
modifies the enzyme to abolish its activity, whereas a reversible inhibitor (I) forms dynamic
complex(es) with the enzyme (E) or the enzyme substrate complex (ES) by reducing the rate of the
enzymatic reaction (see Table 2).
Table 2. Types of Enzyme Inhibitions

WORKSHOPS

1. An enzymatic assay was carried under two different sets of conditions out using a pure
substrate S. The results are tabulated below.

a. Plot the data using the Lineweaver-Burke plot

b. Calculate the values of Vmax and Km for both sets of conditions
c. Suggest possible reasons why the two sets of results might be different.
 Prcticas de Bioqumica. Programa de Biologa.
Daro Mndez Cuadro, Ph D.

2. The initial rates at various substrate concentrations for an enzyme-catalyzed reaction are as
follows:

(a) Does this reaction follow MichaelisMenten kinetics?

(b) Calculate the value of Vmax of the reaction.
(c) Calculate the KM value of the reaction.
(d) Calculate the initial rates at S= 5x10-5M and S= 3x10-1M.
(e) What is the total amount of product formed during the first 3 min at S= 7.2 x10-5M?
(f) How would an increase in the enzyme concentration by a factor of 2 affect each of the
following quantities: KM; Vmax, and v0 (at S= 5x10-5M)?

3. Initial rates of a hydrolase (10.0 nM)-catalyzed reaction are measured and tabulated in table 3.
Evaluate the kinetic parameters of this Uni-substrate reaction. Calculate the turnover number
of the enzyme.
Table 3.

4. A Biologist measured the initial rate of an enzyme-catalyzed reaction in the absence and
presence of inhibitor A and, in a separate procedure, inhibitor B. In each case, the inhibitors
concentration was 8.0 mM. The following data were obtained:

(a) Determine the values of KM and Vmax of the enzyme.

(b) Determine the type of inhibition imposed by inhibitors A and B, and calculate the value of
KI in each case.
 Prcticas de Bioqumica. Programa de Biologa.
Daro Mndez Cuadro, Ph D.

5. Initial rates of an esterase-catalyzed reaction in the absence and presence of 0.10 mM each of
inhibitors I and J are measured and tabulated below. Evaluate the kinetic and inhibition
parameters of this Uni-substrate reaction.

6. Rate measurements were made on a food enzyme with and without the presence of an
inhibitor with the following results

Inhibitor concentration, [I] = 20 mM

a. Use either the Eadie-Hofstee or the Hanes plot to determine the type of inhibition.
b. Determine Km and V0 for the enzyme without inhibition and the inhibitor parameter, KI.
c. What further tests would be necessary to improve the accuracy of estimate of KI?
d. How would you calculate KI in this case?

7. The initial rates (v in M/min) of liver alcohol dehydrogenase-catalyzed ethanal reduction are
measured in the presence of pyrazole as an inhibitor at the constant concentration of NADH
(0.02 M) and the constant concentration of ethanal (2.0 mM), respectively. Propose respective
inhibition types and estimate their inhibition constants.
 Prcticas de Bioqumica. Programa de Biologa.
Daro Mndez Cuadro, Ph D.

8. The active-site-directed inhibition of enzymes has been an important research topic in

pharmaceutical drug design. An early development of anti-cancer agents involved inhibitions
of dihydrofolate reductase and thymidylate synthetase. Search enzyme resource sites for
kinetic data (turnover number, Km and Ki) of these two enzymes, as example
http://www.expasy.ch/enzyme/ and http://www.brenda.uni-koeln.de/.
9. The catalytic residues of serine proteases such as chymotrypsin generally involve catalytic
triad of Asp, His, and Ser residues, for example:

Search the Enzyme Structure Database for -chymotrypsin active site (by the aid of the active-site-
modified enzyme or active-site-specific inhibitorenzyme complex) to identify and depict (save
pdb file) the catalytic triad of -chymotrypsin.

Questions
What are the isozymic? Give an example.
What are allosteric enzymes?
How are the progress curves? What are advantages and disadvantages?

References and links

Stan Tsai. Dynamic Biochemistry: Enzime Kinetics. An Introduction to Computational
Biochemistry. 2002.
Donald Voet; Judith Voet. Biochemistry. 2011. John Wiley sons, inc.
Alejandro G. Marangoni. Enzyme kinetics. A Modern Approach. 2003 by John Wiley & Sons,
Inc. All rights reserved.
Hans Bisswanger. Enzyme Kinetics. Principles and Methods. 2008 WILEY-VCH Verlag GmbH &
Co. KGaA, Weinheim.
Sharon Walker and David Mcmahom. Biochemistry Demystified. 2008 by The McGraw-Hill
Companies.
Enzyme Kinetics: Behavior and Analysis of Rapid Equilibrium and Steady-State Enzyme Systems
 Prcticas de Bioqumica. Programa de Biologa.
Daro Mndez Cuadro, Ph D.

http://www.expasy.ch/enzyme/
http://www.biochem.ucl.ac.uk/bsm/enzyme/index.html
http://www.gebine.ad.jp/dbget/ligand.html
http://www.brenda.uni-koeln.de/
http://wit.mcs.anl.gov/EMP/
http://www.ensam.inra.fr/cholinesterase/
http://www.bi.bbsrc.ac.uk/Merops/Merops.htm
http://delphi.phys.univ-tours.fr/Prolysis
http://afmb.cnrs-mrs.fr/_pedro/CAZY/db.html
http://rebase.neh.com/rebase/rebase.html
http://www.mbio.ncsu.edu/RnaseP/home.html
http://pkr.sdsc.edu
http://PlantP.sdsc.edu
http://rose.man.poznan.pl/aars/index.html
http://metallo.scripps.edu/
http://bmbsgi11.leads.ac.uk/promise/
http://www.ucshc.edu/alcdbase/aldhcov.html
http://www.nal.usda.gov/fnic/foodcomp/
http://qcg.tran.wau.nl/local/pdhc.htm