Enzyme activation intermediate forms during catalysis

As the first committed step in the biosynthesis of AMP from IMP, AMPSase plays a central role in de novo purine nucleotide biosynthesis. A 6-phosphoryl-IMP intermediate appears to be formed during catalysis, and kinetic studies of E. coli AMPSase demonstrated that the substrates bind to the enzyme active sites randomly. With mammalian AMPSase, aspartate exhibits preferred binding to the E GTPTMP complex rather than to the free enzyme. Other kinetic data support the inference that Mg-aspartate complex formation occurs within the adenylosuccinate synthetase active site and that such a... 

Though, in fact, according to Eq. (12) there may be present both the forms [LHM] and [LM], it is also possible to infer that there exist such cases (e.g., intermediate stages during catalysis and chemisorption) when only one of them is of importance. As an example the relationships of the Eq. (14) type describe the dependence of the activity of enzymes (including immobilized ones) on pH [11,34]. Equations (13) can be written as ... 

Much earlier studies on EPSP synthase with radiolabeled substrates suggested the existence of an enzyme intermediate. ° ° There were two plausible mechanisms as illustrated in Scheme 4 (1) a tetrahedral intermediate formed by protonation of the C-3 carbon of PEP, and subsequent attack of the 5 -OH of shikimate-3-phosphate (S3P) to form an intermediate that was not covalently bound to the enzyme as illustrated in the scheme (pathway a), and a covalent intermediate involving a protonated form of PEP that becomes covalently attached to the enzyme through an active site nucleophile during catalysis (pathway b). 

Paracatalytic enzyme reactions on their part may entail paracatalytic enzyme modifications. This new mode of catalysis-linked modification is due to intramolecular reactions of unusual metastable intermediates formed during the paracatalytic reactions. It appears to afford an approach to specific active-site-directed labeling of certain enzymes. The present article describes the principal features of paracatalytic enzyme modification accompanying the oxidation of enzyme-substrate carbanion intermediates, discusses its differentiation from other substrate-dependent and catalysis-linked modifications, and concludes with an account of detailed modification procedures for several enzymes. 

This phosphotransferase [EC 2.7.2.1] catalyzes the thermodynamically favored phosphorylation of ADP to form ATP Aeq = [ATP][acetate]/ [acetyl phosphate] [ADP] = 3000). GDP is also an effective phosphoryl group acceptor. This enzyme is easily cold-denatured, and one must use glycerol to maintain full catalytic activity. Initial kinetic evidence, as well as borohydride reduction experiments, suggested the formation of an enzyme-bound acyl-phosphate intermediate, but later kinetic and stereochemicaT data indicate that the kinetic mechanism is sequential and that there is direct in-line phosphoryl transfer. Incidental generation of a metaphosphate anion during catalysis may explain the formation of an enzyme-bound acyl-phosphate. Acetate kinase is ideally suited for the regeneration of ATP or GTP from ADP or GDP, respectively. 

Whereas the mechanism of the C2-H deprotonation of ThDP has been shown to be identical in all ThDP-dependent enzymes investigated, the following steps in catalysis of the different enzymes require different protonation and deprotonation reactions of the intermediates formed along the process. In order to identify side chains involved in proton transfer steps, the distribution of reaction intermediates during catalysis of any wild type enzyme can be compared with that of active site mutant enzymes. Rate constants for single steps in catalysis can be calculated from... 

Basic to protease catalyzed oligopeptide synthesis is equilibrium- or thermodynamic control to direct reversal of proteolysis . Difficulties encountered include low reaction rates, high stoichiometric amounts of enzyme, and the need to apply direct approaches to shift the reactions towards formation of desired products. Reaction conditions that lead to product precipitation or extraction increase efficiency of the reverse reaction. Kinetically controlled syntheses has proved useful for serine and cysteine proteases that form activated acyl enzyme intermediates during catalysis. This approach generally involves use of activated acyl moieties, such as esters, as donor components which significantly accelerate the reaction rate. This study makes use of principles from both kinetic and thermodynamically controlled reactions in that, reactants are activated by formation of esters and products precipitate fi om reactions. 

Mechanism-based CYP inhibition or irreversible inhibition, involves permanent inactivation of CYP enzymes during catalysis, where reactive intermediate(s) are formed, leading to apoprotein or heme-ion center modification. Typical characteristics of mechanism-based enzyme inhibition include time-dependent loss of enzyme activity, a rate of inactivation generally following saturation kinetics, enzyme activity that cannot be recovered after... 

The contribution of Menz et al is welcomed as providing essential information for the difficult task still ahead of satisfactorily correlating structure with events of substrate binding, covalent catalysis, and product release. For this task, it needs to be recognized that forms revealed by X-ray analysis of static inhibited enzymes may not appear as such during active catalysis, even though they are likely representative of most intermediate forms. ... 

Many enzymes form covalent bonds with their substrates during catalysis, such as the acyl-enzyme intermediate in carboxyl ester hydrolysis (Scheme 2.1) or the glycol monoester intermediate in epoxide hydrolysis (Scheme 2.85). Despite the covalent enzyme-substrate bond, such species are metastable and should be regarded as activated intermediates . Some enzymes utilize cofactors, such as... 

Studies aimed at identifying the active site of an enzyme (cf. 2.4.1.1) have shown that, during catalysis, a number of enzymes bind the substrate by covalent hnkages. Such covalent linked enzyme-substrate complexes form the corresponding products much faster than compared to the reaction rate in a non-catalyzed reaction. Examples of enzyme functional groups which are involved in covalent bonding and are responsible for the transient intermediates of an enzyme-substrate complex are compiled in Table 2.8. Nucleophilic catalysis is dominant (examples 1-6, Table 2.8), since amino acid residues are present in the active site of these enzymes, which... 

APases are widely distributed in prokaryotes as well as in eukaryotes (1) . All purified enzymes are dimeric Zn(lI)-metalloenzymes with an active-site Ser which forms a covalent phosphoserine intermediate during catalysis (2). The molecular sizes range from 80 to 220 kDa per dimer, and mammalian enzymes are generally... 

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