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...

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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...

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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...

Ping Pong Mechanisms

Parallel lines in double reciprocal plots usually mean that a substrate reacts with the enzyme and produces a product independently of the presence of another substrate. In the two-substrate case, scheme 2-7 is the ping pong bi bi mechanism. In this mechanism, the leading substrate A reacts with the enzyme, changing it to a chemically different form designated F in scheme 2-7, and this process leads to the formation and release of the product P. The reaction of E F may involve an enzymatic...

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...

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...

Classes of Ribonucleotide Reductases

We introduced a radical mechanism for the reduction of ribonucleotides in chapter 4, fig. 4-6 in connection with the adenosylcobalamin-dependent RNR. In that case, adenosyl-cobalamin initiates the formation of a thiyl radical in the active site Licht et al., 1996 . The original observation of thiyl radical formation in the adenosylcobalamin-dependent RNR is extended to all other RNRs, but this takes place by different mechanisms at the active sites of the other classes of RNRs Stubbe and van...

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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...

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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...

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...