Enzymes catalyzed condensation study

To make QM studies of chemical reactions in the condensed phase computationally more feasible combined quantum me-chanical/molecular mechanical (QM/MM) methods have been developed. The idea of combined QM/MM methods, introduced first by Levitt and Warshell [17] in 1976, is to divide the system into a part which is treated accurately by means of quantum mechanics and a part whose properties are approximated by use of QM methods (Fig. 5.1). Typically, QM methods are used to describe chemical processes in which bonds are broken and formed, or electron-transfer and excitation processes, which cannot be treated with MM methods. Combined QM and MM methods have been extensively used to study chemical reactions in solution and the mechanisms of enzyme-catalyzed reactions. When the system is partitioned into the QM and MM parts it is assumed that the process requiring QM treatment is localized in that region. The MM methods are then used to approximate the effects of the environment on the QM part of the system, which, via steric and electrostatic interactions, can be substantial. The... 

The second enzyme in the sequence studied was KD0-8-phosphate synthase. This enzyme was purified to homogeneity (24). This enzyme catalyzes the condensation of D-arabinose-5-phosphate and PEP to yield KD0-8-phosphate and inorganic phosphate. One can assay this irreversible reaction either by measuring the formation of KD0-8-phosphate or the release of P. (2Jj). The latter is the method of choice, since a number of analogues were found to interfer with the thiobarbituric acid assay. The enzyme has an apparent K for PEP of 6 x 10 M and an apparent K for D-arabinose-5-phospftiate of 2 x 10 M. m... 

Most known thiamin diphosphate-dependent reactions (Table 14-2) can be derived from the five halfreactions, a through e, shown in Fig. 14-3. Each half-reaction is an a cleavage which leads to a thiamin- bound enamine (center. Fig. 14-3) The decarboxylation of an a-oxo acid to an aldehyde is represented by step h followed by fl in reverse. The most studied enzyme catalyzing a reaction of this type is yeast pyruvate decarboxylase, an enzyme essential to alcoholic fermentation (Fig. 10-3). There are two 250-kDa isoenzyme forms, one an tetramer and one with an (aP)2 quaternary structure. The isolation of a-hydroxyethylthiamin diphosphate from reaction mixtures of this enzyme with pyruvate provided important verification of the mechanisms of Eqs. 14-14,14-15. Other decarboxylases produce aldehydes in specialized metabolic pathways indolepyruvate decarboxylase in the biosynthesis of the plant hormone indole-3-acetate and ben-zoylformate decarboxylase in the mandelate pathway of bacterial metabolism (Chapter 25). Formation of a-ketols from a-oxo acids also starts with step h of Fig. 14-3 but is followed by condensation with another carbonyl compound in step c, in reverse. An example is decarboxylation of pyruvate and condensation of the resulting active acetaldehyde with a second pyruvate molecule to give l -a-acetolactate, a reaction catalyzed by acetohydroxy acid synthase (acetolactate synthase). Acetolactate is the precursor to valine and leucine. A similar ketol condensation, which is catalyzed by the same S5mthase, is... 

These enzymes catalyze preequilibrium proton exchange at the nucleophilic carbon center at a rate consistent with the intermediate involvement of the conjugate base in the condensation reaction (6). The reaction is formally electrophilic substitution of a carbonyl carbon for a proton at the a-carbon atom of the enamine. Stereochemical studies have shown that the proton and carbonyl bind to the same face of the enamine carbon (carbanionic center) (Scheme 7) (7). 

The enzyme catalyzes the condensation of phosphoenolpyruvate and shikimate 3-phosphate to yield 3-phospho-5-enolpyruvoylshikimate (PEPS) [Fig. 2 (6)]. PEPS synthase has been detected in mung bean seedlings (Koshiba, 1978b) properties of the enzyme were not studied, however. The enzyme was detected in bacteria and characterized as a freely reversible reaction (Levin and Sprinson, 1964). 

Aldolases have been studied as catalysts for monosaccharide synthesis for nearly 40 years. The best studied member of the group is a fructose-1,6-diphosphate (FDP) aldolase from rabbit muscle (RAMA, E.C. 4.1.2.13) (20), In vivo, this enzyme catalyzes the reversible condensation of D-glyceraldehyde-3-phosphate and dihydroxy acetone phosphate (DHAP) to generate FDP (Scheme 1). In the synthetic direction, the enzyme catalyzes the formation of two new stereogenic centers with absolute stereospecificity the stereochemistry of the new vicinal diol is always D-... 

Table I. Enzyme-catalyzed condensation study of trimethylsilanol after six days...

Trypsin preferentially catalyzed the condensation of trimethylsilanol under mild conditions (87% HMDS). Subs tial condensation of trimethylsilanol was not observed in the negative control, non-specific protein (i.e. BSA, y-globulins), small molecule (i.e. CaCl2, imidazole, N-methylimidazole), and polypeptide (i.e. poly-L-lysine) reactions in comparison to the raw material (< 1% HMDS). Based on an inqjurity study with small molecule inhibitors (23), the exceptional activity of trypsin and a-chymotrypsin observed in the original enzyme-catalyzed condensation study was soley due to a tryptic impurity. The tertiary... 

Micellization of a copolymer, that may be considered as a comb-graft copolymer was studied by Watterson and co-workers [174]. This copolymer, with C10-C12 alkyl side chains, synthesized through enzyme-catalyzed condensation reaction, is of the following structure ... 

Reactions 35 and 36 have been shown to be catalyzed by different enzyme fractions and the intermediate has been isolated and partially purified. Earlier reports had indicated that reaction 36 was a hydrolytic step resulting in the formation of arginine plus malic acid. However, recent studies indicate that the formation of malic acid was the result of the presence of fumarase in the enzyme preparation. Of considerable interest is the report that a compound similar to, if not identical with, the end product of reaction 35 is enzymatically formed from arginine plus fumaric acid by extracts of plant and animal tissues, and microorganisms. In this connection it has been reported that one of the components of the condensing enzyme system (reaction 35) is present in yeast extracts as well as in liver preparations. Although ATP is required for synthesis of the intermediate from citrulline plus aspartic acid, it is not needed for the synthesis from arginine plus fumaric acid. 

Thiamine, one of the oldest and best known vitamins in some respects, is stUl essentially unknown as regards its biogenesis. Neither the origin of the pyrimidyl portion nor of the thiazole ring has been studied as yet by modem biochemical methods. It is dear only that the thiamine molecule can be synthesized both in vitro and in vivo by an enzymically catalyzed condensation of the pyrimidyl and thiazole moieties. 

Purified enzyme from Escherichia coli has been studied for its reactivity tvith homologous five-carbon phosphosugars to yield KDO derivatives, and tvith modified nucleophiles. Thus, the stereochemically distinct fiuoro-analogs of PEP (Z)- and ( )-31 could be separately condensed vith D-erythrose 4-phosphate to yield, stereospecifically, the corresponding (3S)-and (3R)-configured, fiuoro-substituted DAHP derivatives 32 and 33, respectively (Figure 5.18) [127]. This provides direct evidence that the enzyme catalyzes si face addition of the C-3 of PEP to the rc face of the acceptor aldehyde. 

Tewfik and Stumpf (347) found aldolase to be present in all plant species and tissues studied. Stumpf (338) purified pea aldolase and found no requirement for divalent cations this enzyme thus resembles that found in muscle, rather than that in yeast (381), bacteria (28,124), and molds (189). Like aldolase from muscle, the pea enzyme will catalyze the condensation of dihydroxyacetone phosphate with any one of a number of aldehydes. The condensation reactions catalyzed by pea aldolase have been studied by Hough and Jones (185 and Gorin and Jones (137). 

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