Creatine Kinase

Creatine phosphate serves as a cellular storage species of high-energy phosphate that is readily available for the formation of ATP from ADP by the action of creatine kinase (EC 2.7.3.2). Creatine phosphate is a high-energy phosphate (AG' -10 kcal mol-1 for hydrolysis) by virtue of being a phosphoramidate. The reversibility of the reaction of creatine kinase (eq. 10-5), allows creatine phosphate to be formed whenever the concentration of ATP is high, and it makes creatine phosphate an efficient...

TPP in Decarboxylation

E1 catalyzes the TPP-dependent decarboxylation of pyruvate by a mechanism related to the action pyruvate decarboxylase discussed in chapter 8 and illustrated in fig. 18-4. TPP-dependent decarboxylation begins with ionization of the thiazolium-C2(H) to form the reactive ylid (see chap. 3). This is brought about by a glutamate residue, Glu571 in the E. coli enzyme, in a process mediated by N1 and N4' of the pyrimidine ring, as shown in fig. 18-4A. The ylid reacts as a nucleophile with the...

Inhibition Patterns

The primary plots of steady-state kinetic data often do not allow an unambiguous decision regarding the kinetic mechanism in multisubstrate reactions. We have seen that eq. 2-10 is the rate law for three recognized mechanisms. Inhibition studies can distinguish among the mechanisms. Products combine with the enzyme and tend to reverse the reaction. A dead-end inhibitor may resemble a substrate or product but not react. An inhibitor can be a substrate molecule when the substrate concentration is...

Scope and Limitations

More than a hundred catalytic antibodies have been described that catalyze a variety of reactions. The most striking examples are those that catalyze aldol and Diels-Alder reactions. The aldolase antibodies function by a mechanism similar to that of type I aldolases, with a lysyl residue at the binding site that forms an iminium intermediate analogous to that of aldolase in fig.1-12 (Barbas et al., 1997 Sinha et al., 1998 Wagner et al., 1995). Unlike aldolase, the aldolase antibody displayed...

Structure of Yeast Pyruvate Decarboxylase

Structure Pyruvate Decarboxylase

Several structures of PDC from yeast and bacteria reveal much about the chemical environment at the active site and the structure of TPP at this site (Aijunan et al., 1996 Dobritzsch et al., 1998 Dyda et al., 1993). The structure of yeast PDC in fig. 8-3 reveals the complexities of a tetrameric multidomain protein with several binding sites. In addition Fig. 8-3. Pyruvate decarboxylase from yeast is a homotetrameric protein that is subject to allosteric regulation. The four subunits of the...

I[A

This simplified scheme does not include the E.Q or F.P complexes, but it sufficies to give a valid equation the inclusion of the product complexes gives a hexagonal instead of a square pattern, and the resulting kinetic equation has the same form with more rate constants. The writing of the basic scheme in a cyclic pattern is step 1 of the King-Altman derivation. In step 2, all possible patterns are drawn with one side of each loop missing. In the case of the square pattern, there is a single...

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...

O

Decarboxylase Mechanism

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...

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...

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...

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...

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...