Chapter 6

Initial-Rate Kinetics of Multi-Substrate

Enzyme-Catalyzed Reactions
In enzyme kinetics, the terms two-substrate and three- catalyze three-substrate reactions, such as those catalyzed
substrate mechanisms refer to those, for which the reaction by phenylalanine-tRNA synthase (Reaction: Phenylalanine þ
rate v depends on the concentrations of two and three tRNAPhe þ MgATP2 # Phenylalanyl-tRNAPhe þ AMP þ
substrates, respectively. Most enzymes actually catalyze PPi), glutamine synthetase (Reaction: L-Glutamate þ NH3
reactions involving two or more substrates, and, with the þ MgATP2 # L-Glutamine þ MgADP þ Pi), and
obvious exception of isomerases, many ‘‘one-substrate’’ 3-hydroxy-3-methylglutaryl-CoA reductase (Reaction: (S)-
enzymes actually have two substrates. Examples are prote- 3-hydroxy-3-methylglutaryl-CoA þ 2 NADPH # (R)-
ases and peptidases (Reaction: Polypeptidemþn þ H2O # mevalonate þ CoA þ 2 NADPþ). Other multi-substrate
Fragmentm–COOH þ NH2–Fragmentn, where the sub- enzymes, such as those utilizing pyridoxal 5-P, heme or
scripts m and n indicate the number of residues). At 55.5 M, biotin, employ covalently bound coenzymes, which cycles
the water concentration remains unchanged throughout between two different covalently modified forms. Exam-
the course of reaction, and water is not a kinetically ples include transaminases (Reaction: Amino-Acid1 þ
discernible substrate. In the reverse direction, peptidases Oxo Acid2 # Amino-Acid2 þ Oxo Acid1), catalase
clearly require two substrates, and the same is true for other (Reaction: 2 H2O2 / O2 þ 2 H2O), and pyruvate
hydrolases (e.g., phosphatases, esterases, glycosidases, etc.) carboxylase (Reaction: Pyruvate þ CO2 þ GTP #
as well as certain lyases (e.g., Fumarase Reaction: Malate # Oxaloacetate þ GDP þ Pi). These behave as essential
Fumarate þ H2O). Tightly bound cofactors that fail to cofactors, coenzymes, and prosthetic groups.
dissociate after each catalytic cycle or are released only The order of substrate binding determines the kinetic
after multiple catalytic cycles are likewise kinetically properties of multi-substrate reactions to such an extent
indiscernible as a second substrate. An example is UDP- that, to investigate other important aspects of catalysis, the
galactose 4-epimerase, which contains an extremely substrate binding order must first be determined, along with
tightly bound NADþ molecule at its active site. This their respective composite kinetic parameters Km, Vm, and
redox coenzyme participates in a hydride-transfer reaction Vm/Km. Compulsorily ordered kinetic mechanisms suggest
from UDP-galactose to generate a UDP-4-ketohexopyr- the likely involvement of substrate-induced conformational
anoside intermediate that is then reduced stereospecifi- changes. The kinetic mechanism for substrate addition and
cally by the same enzyme-bound NADH to form product release also greatly influences the way in which
UDP-glucose. There are many reactions that have one reaction products inhibit a given enzyme. The same is true
substrate in one direction and two in the other. The C–C for the activation or inhibition of an enzyme by naturally
bond-cleaving enzyme aldolase, for example, utilizes occurring and synthetic agents. Determination of the kinetic
a single substrate (fructose 1,6-bisphosphate) in the mechanism of multi-substrate enzymes is therefore a major
direction of glycolysis and two substrates (glyceralde- endeavor, one that has received considerable theoretical and
hyde-3-P and dihydroxyacetone-P) in the direction of practical attention. This chapter focuses on the initial-rate
gluconeogenesis. Finally, because nearly all enzyme behavior of multi-substrate enzyme-catalyzed reactions.
studies are conducted in buffered solutions, protons are Other kinetic properties are treated in Chapter 7, and
not normally treated as substrates. various modes of inhibitor action are described in Chapter
Many enzymes employ reversibly bound phosphoryl- 8. Later chapters describe their isotope exchange behavior,
and nucleotidyl-donors (e.g., MgATP2 and MgGTP2), kinetic isotope effects, and fast reaction kinetics.
redox coenzymes (e.g., NADH and FADH), and acyl-
transfer coenzymes, like Acetyl-Coenzyme A. These
behave in every respect as true substrates. Examples include 6.1. BISUBSTRATE KINETIC MECHANISMS
hexokinase (Reaction: Glucose þ MgATP2 # Glucose 6-
P þ MgADP) and lactate dehydrogenase (Reaction: Lactate þ As first suggested by Segal, Kachmar and Boyer (1952),
NADþ # Pyruvate þ NADH þ Hþ). Other enzymes valuable information on the kinetic mechanisms of

Enzyme Kinetics
Copyright Ó 2010, by Elsevier Inc. All rights of reproduction in any form reserved. 335