Oxyanion intermediates

The family of serine proteases has been subjected to intensive studies of site-directed mutagenesis. These experiments provide unique information about the contributions of individual amino acids to kcat and KM. Some of the clearest conclusions have emerged from studies in subtilisin (Ref. 9), where the oxyanion intermediate is stabilized by t>e main-chain hydrogen bond of Ser 221 and an hydrogen bond from Asn 155 (Ref. 2). Replacement of Asn 155 (e.g., the Asn 155— Ala 155 described in Fig. 7.9) allows for a quantitative assessment of the effect of the protein dipoles on Ag. ... 

Orbitals, atomic, see Atomic orbitals Orbitals, molecular, see Molecular orbitals Orbital steering mechanism, 220-221 Oxyanion intermediates, 172,181,185,210 Oxyanion hole, 181... 

KINETIC ISOTOPE EEEECTS 5-OXOPROLINASE Oxyanion intermediate,... 

The catalytic cycle. Figure 12-11 depicts the generally accepted sequence of reactions for a serine protease. If we consider both the formation and the subsequent hydrolysis of the acyl-enzyme intermediate with appropriate oxyanion intermediates, there are at least seven distinct steps. As indicated in this figure, His 57 not only accepts a proton from the hydroxyl... 

NH groups is apparently good only for the tetrahe-drally bonded oxyanion intermediate,262 a structure thought to be close to that of the transition state. The importance of the oxyanion hole for catalysis has been supported by theoretical calculations245 and by the study of mutant enzymes.263 In subtilisin, in which a side chain of asparagine forms part of the oxyanion hole, replacement of Asn with the isosteric Leu causes /ccat to fall by a factor of about 200 while Km is unaffected 264 It is also of interest that thiono ester substrates, in which the C=0 of an oxygen ester has been replaced by C = S, bind with normal affinity to chymotrypsin but are not hydrolyzed at significant rates.265... 

The formation of the oxyanion intermediate during serine protease action is also supported by the existence of tetrahedral forms of enzymes inhibited by substratelike aldehydes. The -OH group of Ser 195 can add to the carbonyl group to form hemiacetals. For example, a 13C-enriched aldehyde whose carbonyl carbon had a chemical shift of 204 ppm gave a 94 ppm resonance as it formed the tetrahedral hemiacetal with one of the inhibitory aldehydes, N-ace tyl-i -Len-i-Leu-L-arginal... 

The probable mechanism of action of chymotrypsin. The six panels show the initial enzyme-substrate complex (a), the first tetrahedral (oxyanion) intermediate (b), the acyl-enzyme (ester) intermediate with the amine product departing (c), the same acyl-enzyme intermediate with water entering (d), the second tetrahedral (oxyanion)... 

Stabilization of enolate anions generated from abstraction of a proton a to a carboxylate Hydrolysis, phosphoryl group transfer via hydrolytic nucleophilic substitution Stabilization of diverse oxyanion intermediates via metal-assisted catalysis Schiff base dependent formation of an electron sink ... 

Stabilization of oxyanion intermediates derived from thioesters... 

The mechanism of phosphonate anion (135) addition to carbonyl derivatives is similar to the phosphonium ylide addition however, there are several notable features to these anion additions that distinguish the reactions fix)m those of the classical Wittig. The addition of the anion gives a mixture of the erythro (136 and 137) and threo (139 and 140) isomeric p-hydroxyphosphonates (Scheme 24). In the case of phosphine oxides, the initial oxyanion intermediates may be trapped. The anion intermediates decompose by a syn elimination of phosphate or phosphinate to give the alkene. The elinunation is stereospecific, with tile erythro isomer producing the ci.r-alkene (138), and the threo addition adduct producing the... 

Scheme 11.1 General acid and base catalysis of deamidation. The tetrahedral oxyanion intermediate is inferred to be the transition state.

The proposed importance of hydrogen bonds in providing significant differentia] stabilization of oxyanion intermediates was quickly challenged. Kluger and Guthrie... 

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