Coenzymes II Metallic Coenzymes

The original coenzymes were small organic molecules that activated enzymes and participated directly in catalyzing enzymatic reactions. Most of them were derived from vitamins and were known as biologically activated forms of vitamins such as niacin, riboflavin, thiamine, and pyridoxal. Heme was in a separate category, perhaps because of its widespread biological role as an oxygen carrier, and because it was not a vitamin, it was not widely regarded as a coenzyme. However, heme was clearly an...

Enzymatic Rate Enhancement and Transition State Binding

Accepting transition state theory as a basis for understanding reaction kinetics, it can be shown that an enzyme (or any catalyst) can catalyze a reaction by binding the transition state more tightly than the ground state. This concept made its appearance early in the study of enzymes (Pauling, 1948). It is a straightforward and reasonable concept because of the fact that a binding interaction should be stabilizing therefore, binding the transitionstate should stabilize it, thereby increasing...

Isotope Exchange at Equilibrium

We have discussed isotope exchange between a substrate and product (e.g., A*-P*) in the absence of the second substrate in connection with verifying the ping pong bi bi mechanism. Such exchanges do not occur in sequential mechanisms because substrate-product interconversion requires the presence of all substrates and products. However, when the substrates and products are present, the kinetics of substrate-product isotope exchange at equilibrium can distinguish between random and ordered...

O

Pyruvoyl-enzymes to be discovered (Recsei and Snell, 1984 van Poelje and Snell, 1990). Pyruvoyl-enzymes catalyze a-decarboxylation of amino acids, similar to many PLP-dependent enzymes, and they include histidine, S-adenosylmethionine, phosphatidyl serine, and aspartate a-decarboxylases. Pyruvoyl-enzymes are found in both prokaryotes and eukaryotes. d-Proline reductase is also a pyruvoyl-enzyme (Hodgins and Abeles, 1967). The pyruvoyl moiety in histidine decarboxylase arises in a self-cleavage...

PALA and Aspartate Transcarbamylase

An early example of the binding of a two-substrate analog was the inhibition of aspartate transcarbamylase (ATCase) by N-(phosphonoacetyl)-L-aspartate (PALA), which is shown in structure 5-1. ATCase is described in chapter 2. ATCase catalyzes the reaction of aspartate with carbamyl-P to produce carbamylaspartate in the first step of pyrimidine biosynthesis. ATCase binds PALA very tightly at its active site in competition with its substrates (Collins and Stark, 1971). PALA is a very potent...

Role of Ile16

To comprehend the identification of Ile16 as an ionizing group governing pH dependence, it is necessary to understand the origin of the B chain. Mature a-chymotrypsin consists of chains A, B, and C that arise from proteolytic processing of chymotrypsinogen, the inactive proenzyme form and primary translation product. Activation of chymotrypsinogen in vitro by the action of trypsin and activated chymotrypsin leads to the species depicted in fig. 6-2. Trypsin catalyzes peptide hydrolysis at sites...

Glu35 and Asp52 in Lysozyme

Chemical modification of lysozyme provides several enduring lessons in enzyme science. Lysozyme catalyzes the hydrolysis of bacterial cell walls by cleavage between N-acetyl-glucosaminyl residues, and it is the first enzyme structure to be obtained by x-ray crystallography. The structure revealed the presence of Glu35 and Asp52 in the active site cleft. Exhaustive chemical modification of the carboxylic amino acid side chains by the method of water-soluble carbodiimide activation coupled with...

Ordered Sequential Mechanisms

As in the case of single-substrate reactions, the substrate-binding steps are often not at equilibrium, and substrate and product dissociation are not much faster than the interconversion of ternary complexes. In these cases, substrate binding must be described in terms of rate constants, as in the Briggs-Haldane formulation, not dissociation constants. Nevertheless, the experimental rate equation often has the same form as eq. 2-10 for the equilibrium random mechanism. The ordered bi bi...

Decarboxylation and Carboxylation

Decarboxylation is an essential process in catabolic metabolism of essentially all nutrients that serve as sources of energy in biological cells and organisms. The most widely known biological process leading to decarboxylation is the metabolism of glucose, in which all of the carbon in the molecule is oxidized to carbon dioxide by way of the glycolytic pathway, the pyruvate dehydrogenase complex, and the tricarboxylic acid cycle. The decarboxylation steps take place in thiamine pyrophosphate...

Kinetic and Equilibrium Isotope Effects

Isotope effects can contribute important information in the mechanistic analysis of enzymatic catalysis. An isotope effect is any effect on a reaction that is induced by the substitution of a heavy isotope into a reacting molecule. isotope effects may be induced on either the rate of or the equilibrium constant for a reaction. Effects on the rate are kinetic isotope effects, and effects on the equilibrium constant are equilibrium isotope effects. Most often the kinetic isotope effects provide...

Hal

Hypothetical mechanisms for the action of histidine ammonia-lyase. The mechanisms differ in the function assigned to methylidene imidazolone. In mechanism 1, the amino group of histidine undergoes nucleophilic addition, and its reactivity as a leaving group in the resulting complex drives its elimination. An enzymatic base simultaneously accepts the benzyllike P-proton. In mechanism 2, the imidazole group of histidine undergoes nucleophilic addition to the coenzyme, and this process...

Chemistry of ATP Synthase and the Binding Change Mechanism

As shown in biochemical studies, ATP synthase has preferential binding sites for MgATP and MgADP. Moreover, the kinetic studies of exchange reactions during hydrolysis by F1-ATPase and ATP synthesis by ATP synthase and the effects of varying the free nucleotide concentration on the exchange kinetics, provide valuable clues to site-site interactions within the multimeric enzyme. As shown in early radiochemical experiments, F1-ATPase catalyzes the rapid exchange of 32Pi into unreacted ATP, the...

Jvv

Mechanisms for glutaminase action and inactivation by diazonorleucine DON . A The mechanism accounts for the role of the essential cysteine residue in glutaminase action. Cysteine functions as a nucleophilic catalyst, leading to ammonia and a covalent y-glutamyl thioester-enzyme intermediate. Hydrolysis of the y-glutamyl thioester leads to glutamate. B Inactivation of glutaminase by 6-diazo-5-oxo-norleucine DON is a suicide mechanism initiated by the addition of the active site...

Histidine Phosphatases

Histidine Phosphorylation Mechanism

Phosphatases displaying full activity at pH 2.5, with subunit molecular masses of 40 to 60 kDa and dimeric structures, constitute a distinct family. The human lysosomal and prostatic acid phosphatases employ histidine as the nucleophilic catalyst Van Etten, 1982 . Trapping experiments with the substrate p-nitrophenyl 32P phosphate and alkaline denaturation in the steady state leads to a 32P-labeled protein, which on alkaline hydrolysis produces to 81-phosphohistidine Van Etten and Hickey, 1977...

Appendix D Derivation of Steady State Kinetic Equations by the King Altman Method

Enzymatic activities are typically measured under conditions in which the substrate concentrations are much higher than the enzyme concentrations, and this ensures the validity of the steady-state approximation. A typical kinetic mechanism includes several or many enzyme forms, such as enzyme-substrate and enzyme-product complexes or chemically modified enzyme forms, all of which are connected by ligand binding or chemical steps. The number of enzyme forms may be as few as three E, E.S, and E.P...

Concerted Acid and Base Catalysis

Because an enzyme brings reacting groups together in the Michaelis complex, the possibilities for concerted general acid and general base catalysis are maximized. An early model of concerted catalysis in solution was observed in the mutarotation of tetramethyl glucose Swain and Brown, 1952 . The bifunctional catalyst 1-pyridone, the dominant tautomer of 1-hydroxypyridine, was 104 times as effective as an equimolar mixture of phenol and pyridine in catalyzing mutarotation, presumably due to...

Specific Acid and Base Catalysis

A specific acid-catalyzed reaction in water is accelerated by the binding of a proton to a functional group of the substrate. A familiar example is the acid-catalyzed hydrolysis of a peptide. The amide functional group is hardly reactive toward water, which makes it well suited to serve as the basic structural unit in proteins. Its stability is due to delocalization of the nonbonding electron pair, as illustrated in structures 1-1 and 1-2, shown in resonance notation. The charge separated...