Enzyme covalently bound intermediates

The topologically defined region(s) on an enzyme responsible for the binding of substrate(s), coenzymes, metal ions, and protons that directly participate in the chemical transformation catalyzed by an enzyme, ribo-zyme, or catalytic antibody. Active sites need not be part of the same protein subunit, and covalently bound intermediates may interact with several regions on different subunits of a multisubunit enzyme complex. See Lambda (A) Isomers of Metal Ion-Nucleotide Complexes Lock and Key Model of Enzyme Action Low-Barrier Hydrogen Bonds Role in Catalysis Yaga-Ozav /a Plot Yonetani-Theorell Plot Induced-Fit Model Allosteric Interaction... 

This exchange reaction, as well as other predicted exchanges, has been observed.44 Although die exchange criterion of the mechanism is often applied to enzymatic processes, the observation of exchange reactions does not prove the existence of a covalently bound intermediate. Furthermore, enzymes using doubledisplacement mechanisms may not always catalyze the expected exchanges. 

The second mechanism, called the retaining mechanism, forms a covalently bound intermediate through nucleophilic attack of the charged amino acid on the glycosyl bond. A second step, where a water molecule is activated for nucleophilic attack, frees the hydrolysis product from the enzyme and recharges the proton donor (Fig. 9). 

Evidence has been provided for a covalently bound intermediate in a j9-glucosidase mediated glycosyl fluoride hydrolysis. 2-Deoxy-2-fluoro-j8-D-glucopyranosyl fluoride becomes bound to the enzyme and then the 2-fluoro group considerably slows what is normally a fast hydrolysis step to regenerate the enzyme and produce the hydrolysed sugar. The adduct formed between 2-deoxy-2-fluoro- >D-mannopyranosyl fluoride and the enzyme was shown... 

In the chymotrypsiii mechanism, the nitrophenylacetate combines with the enzyme to form an ES complex. This is followed by a rapid second step in which an acyl-enzyme intermediate is formed, with the acetyl group covalently bound to the very reactive Ser . The nitrophenyl moiety is released as nitrophenolate (Figure 16.22), accounting for the burst of nitrophenolate product. Attack of a water molecule on the acyl-enzyme intermediate yields acetate as the second product in a subsequent, slower step. The enzyme is now free to bind another molecule of nitrophenylacetate, and the nitrophenolate product produced at this point corresponds to the slower, steady-state formation of product in the upper right portion of Figure 16.21. In this mechanism, the release of acetate is the rate-llmitmg step, and accounts for the observation of burst kinetics—the pattern shown in Figure 16.21. 

FIGURE 18.5 Schematic representation of types of multienzyme systems carrying out a metabolic pathway (a) Physically separate, soluble enzymes with diffusing intermediates, (b) A multienzyme complex. Substrate enters the complex, becomes covalently bound and then sequentially modified by enzymes Ei to E5 before product is released. No intermediates are free to diffuse away, (c) A membrane-bound multienzyme system. 

Scheme 10.8 Biosynthesis of epothilone. Individual PKS domains are represented as circles and individual NRPS domains as hexagons. Acyl carrier proteins (ACPs) and thiola-tion domains (T) are posttranslationally modified by a phos-phopantetheinyl group to which the biosynthetic intermediates are covalently bound throughout the chain assembly. The thioesterase domain (TE) cyclizes the fully assembled carbon chain to give the 16-membered lactone. Following dehydration of Cl 2—Cl 3 to give epothilones C and D, the final step in epothilone biosynthesis is the epoxidation of the C12=C13 double bond by the cytochrome P450 enzyme P450epol<. KS ketosyn-thase KS(Y) active-site tyrosine mutant of KS AT acyltransfer-ase C condensation domain A adenylation domain ...

The calculations found there was no covalent intermediate in the viral neuraminidase reaction and the intermediate was more likely to be hydroxylated directly. Because there is only a small energy difference between the two options (formation of a covalent bond or direct hydroxylation) Thomas et al. proposed it might be possible to design inhibitors covalently bound to the enzyme. 

Sometimes CYPs can also produce reactive metabolite species that, instead of undergoing the normal detoxification pathway, can act as irreversible CYP inhibitors, thus causing toxicity. Such reactive metabolites that cause CYP inactivation are called MBI and are described in Chapter 9. Mechanism-based enzyme inhibition is associated with irreversible or quasi-irreversible loss of enzyme function, requiring synthesis of new enzymes before activity is restored. The consequences of MBI could be auto-inhibition of the clearance of the inactivator itself or prolonged inhibition of the clearance of other drugs that are cleared by the same isozyme. There may also be serious immunotoxicological consequences if a reactive intermediate is covalently bound to the enzyme. Therefore, screening of new compounds for MBI is now a standard practice within the pharmaceutical industry. 

Fig. 1 Catalytic mechanism of CALB showing an acylation and deacylation step and the formation of a covalently bound acyl-enzyme intermediate bottom right) [16]...

The glycosidases act by two different mechanisms which is revealed by the stereochemistry at the anomeiic centre of the product (McCarter and Withers, 1994). In one type of glycosidases the anomeiic centre is directly attacked by a hydroxide to give a product with inverted stereochemistry at the anomeiic centre. In the other mechanism, the anomeric centre is attacked by the carboxylate group of a glutamic acid residue to form an intermediate in which the carbohydrate moiety is covalently bound to the enzyme similar to in epoxide hydrolases (Figure 2.16) and serine hydrolases. Attack on this intermediate by a nucleophile leads to the net result which is retention of the stereochemistry at the anomeric centre. 

FIGURE 16-17 Biological tethers. The cofactors lipoate, biotin, and the combination of /3-mercaptoethylamine and pantothenate form long, flexible arms in the enzymes to which they are covalently bound, acting as tethers that move intermediates from one active site to the next. The group shaded pink is in each case the point of attachment of the activated intermediate to the tether. 

In the preceding section, four diagnostic tests of affinity labeling were listed (inactivation inhibited by substrates, pH dependence of inactivation similar to that of catalysis, labeled inhibitor covalently bound in 1 1 stoichiometry, and saturation kinetics obeyed). The same criteria may be used to diagnose suicide inhibition. In addition, tests must be made to detect any diffusion of the activated intermediate I into solution. For example, the addition of —SH reagents that rapidly react with electrophiles and hence scavenge them should not slow down the rate of reaction. The suicide inhibitor should not, in any case, react with the thiol at an appreciable rate in the absence of enzyme. 

It is clear that this mechanistic interpretation is not unique, because there is no direct evidence for the proposed intermediates. For example, alternative possible explanations are (1) The amino group in the aminoenzyme may not be covalently bound, but may merely be activated by the enzyme and (2) similarly, the acyl transfer reaction of equation 16.31 could occur by the direct attack of Leu-Tyr-Leu on the enzyme-bound Leu-Tyr-Leu. However, M. S. Silver and S, L. T. James169,170 have proposed a further interpretation, based on the observation that small peptides stimulate the pepsin-catalyzed hydrolysis of other peptides by being first synthesized into larger peptides in a condensation reaction that is the reverse of the hydrolytic step e.g., equations 16.33. The idea of the condensation of two small peptides to give a larger peptide at a rate that is relatively fast compared with hydrolysis of the small peptides is quite reasonable... 

This enzyme is a non-specific phosphomonoesterase that shows maximum activity at pH values greater than 8.569 It also catalyzes the transfer of phosphoryl groups. These reactions involve the formation of a phosphoseryl intermediate and the hydrolyzed substrate. The phosphoenzyme may transfer the phosphoryl group to water or to an acceptor molecule to give a new phosphoester (equations 19 and 20, where E—P represents the covalently bound phosphoenzyme and E-P a non-covalent complex, in which phosphate is coordinated to the zinc). The phosphoenzyme may be formed from either direction. 

A reaction looked at earlier simulates borate inhibition of serine proteinases.33 Resorufin acetate (234) is proposed as an attractive substrate to use with chymotrypsin since the absorbance of the product is several times more intense than that formed when the more usual p-nitrophcnyl acetate is used as a substrate. The steady-state values are the same for the two substrates, which is expected if the slow deacylation step involves a common intermediate. Experiments show that the acetate can bind to chymotrypsin other than at the active site.210 Brownian dynamics simulations of the encounter kinetics between the active site of an acetylcholinesterase and a charged substrate together with ah initio quantum chemical calculations using the 3-21G set to probe the transformation of the Michaelis complex into a covalently bound tetrahedral intermediate have been carried out.211 The Glu 199 residue located near the enzyme active triad boosts acetylcholinesterase activity by increasing the encounter rate due to the favourable modification of the electric field inside the enzyme and by stabilization of the TS for the first chemical step of catalysis.211... 

In Equation 11.13, A = k2k3[E]0[S]0/(k2 + k3) / [S]0 + k3Ks/(k2 + k3), which has the form of a Michaelis-Menten equation, B = [E]0[S]0 /(fe + 3) 2/([S]0+ Km(apparent)), and b is a composite rate constant describing the build-up of the acyl enzme intermediate (or, in the general case, the covalently bound enzyme intermediate). The non-linear plot of [Lg ] against time is shown in Fig. 11.10A for a typical substrate of a-chymotrypsin extrapolation of the linear portion gives the intercept shown which allows evaluation of B. 

The proton donated from the OH group of Ser 195 to His 57 is then donated to the N atom of the scissile bond, cleaving the C-N peptide bond (or the C-O ester bond) to produce the amine and the acyl-enzyme intermediate. The amine is that part of the substrate which follows the scissile bond in the sequence the acyl-enzyme intermediate is the remaining fragment covalently bound to Ser 195. 

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