Ester hydrolysis 130 Histidine

In chymotrypsin and other serine proteases the imidazole moiety of histidine acts as a general base not as a nucleophile as is probably the case in the catalysis of activated phenyl ester hydrolysis by (26). With this idea in mind, Kiefer et al. 40) studied the hydrolysis of 4-nitrocatechol sulfate in the presence of (26) since aryl sulfatase, the corresponding enzyme, has imidazole at the active center. Dramatic results were obtained. The substrate, nitrocatechol sulfate, is very stable in water at room temperature. Even the presence of 2M imidazole does not produce detectable hydrolysis. In contrast (26) cleaves the substrate at 20°C. Michaelis-Menten kinetics were obtained the second-order rate constant for catalysis by (26) is 10 times... 

Considerable effort has been applied to studies of ester hydrolysis catalyzed by imidazoles (76MI40700, 80AHC(27)241). Certainly, 1-acetylimidazole can be made enzymically, probably by the sequence acetyl phosphate + coenzyme A acetylcoenzyme A+phosphate, acetyl-coenzyme A + imidazole l-acetylimidazole+coenzyme A. In addition, the imidazolyl group of histidine appears to be implicated in the mode of action of such hydrolytic enzymes as trypsin and chymotrypsin, thereby engendering further interest in the process of imidazole catalysis. The two pathways which have been found to be involved are general base catalysis and nucleophilic catalysis. In the former (Scheme 26) a basic imidazole molecule can activate a water molecule to attack the ester at the carbonyl carbon, this being followed by the usual sequence of steps as in simple hydroxide ion hydrolysis. At high imidazole concentrations the imidazole molecules may be involved directly. 

Histidine can be prepared from imidazole as in Scheme 117. It is a basic amino acid, the biosynthesis of which appears to involve adenosine 5 -phosphate, ribose phosphate and glutamine (which supplies a nitrogen for the imidazole ring) (Scheme 118). Histidine is one of the most important amino acids and has involvement in such biological processes as ester hydrolysis, acylation and (by virtue of its complexing power with iron) oxygen... 

The catalysis of hydrolysis of carboxylic acid derivatives by weak bases has not been carefully studied until relatively recently. Koshland reported in 1952 the catalysis of acetyl phosphate hydrolysis by pyridine Bafna and Gold (1953) reported the pyridine-catalyzed hydrolysis of acetic anhydride. A short time later the catalysis of aromatic ester hydrolysis by imidazole was demonstrated (Bender and Turnquest, 1957 a, b Bruice and Schmir, 1957). Since that time a large amount of work has been devoted to the understanding of catalyzed ester reactions. Much of the work in this area has been carried out with the purpose of inquiry into the mode of action of hydrolytic enzymes. These enzymes contain on their backbone weak potential catalytic bases or acids, such as imidazole in the form of histidine, carboxylate in the form of aspartate and glutamate, etc. As a result of the enormous effort put into the study of nucleophilic displacements at the carbonyl carbon, a fair understanding of these reactions has resulted. An excellent review is available for work up to 1960 (Bender, 1960). In addition, this subject has been... 

Two attempts have been made to demonstrate an enhancement in the efficiency of the imidazole-catalyzed phenyl ester hydrolysis by using micelles carrying imidazole residues. It was found that micelles formed by myristoyl-L-histidine (MH)... 

At this juncture it is convenient to discuss the catalysis of ester hydrolysis by imidazole. The imidazolyl group of histidine is intimately involved in the mode of action of hydrolytic enzymes, and the two pathways believed to operate, general base catalysis and nucleophilic catalysis, were described in CHEC-I. In the latter, a 1-acetylimidazole is formed as an intermediate. General base catalysis has been proposed in the intramolecular thiolysis of A-propyl-2-thiomethylbenzamide... 

As with peptide hydrolysis, several enzyme systems exist that catalyze carboxylic and phosphoric ester hydrolysis without the need for a metal ion. They generally involve a serine residue as the nucleophile in turn, serine may be activated by hydrogen-bond formation—or even proton abstraction—by other acid-base groups in the active site. The reaction proceeds to form an acyl- or phosphory 1-enzyme intermediate, which is then hydrolyzed with readdition of a proton to the serine oxygen. Mechanisms of this type have been proposed for chymotrypsin. In glucose-6-phosphatase the nucleophile has been proposed to be a histidine residue. ... 

Following the initial observation of stereoselectivity in ester hydrolysis by optically active surfactant histidines, several recent observations have served to reinforce its generality. Yamada and co-workers suggest that both r>- and l-myristoylhistidine show the same selectivity (1.5 1) in mixed micelles with (78) towards N-ethoxycarbonylphenylalanine p-nitrophenyl ester, and thus the... 

Baltzer s group has recently described a fully-synthetic protein that is also capable of hydrolysing p-nitrophenyl esters the polypeptide, which contains 42 amino acids, was designed to fold into a hairpin helix-loop-helix motif that dimerises into a four-helix bundle. The dimer is predicted to present on its surface a shallow reactive site containing several histidine residues. The spectroscopic properties of the peptide are consistent with the predicted folded structure, and the molecule does indeed catalyse ester hydrolysis (and transesterification) more effectively than 4-methylimidazole does. However, there is little substrate selectivity, and not much turnover. The histidine array does not seem to act via general acid-base catalysis, but rather to bind and stabilise ester oxygens in the transition state. We return to this molecule below. 

Ester Hydrolysis. Inspired by evidence that the imidazole ring of histidine residues present in various hydrolytic enzymes is responsible for their proteolytic activities, imidazole itself has been shown to be an excellent catalyst of ester hydrolysis (e.g. eq 2). In intramolecular transesterifications and hydrolyses of 2-hydroxymethylbenzoic acid derivatives, the accelerating role of imidazole is due to its ability to act as a proton transfer catalyst rather than as a nucleophile. ... 

Reymond et al. report the synthesis of peptide dendrimers displaying multiple serine-histidine (diad) residues to catalyze ester hydrolysis reactions [33], Best results where obtained with the fourth generation dendrimer 36. The rate acceleration of the hydrolysis of a fluorescent nonanoyl ester (35) was 140,000-fold more efficient than with 4-melhyl-imidazole as reference catalyst (Scheme 7.7). 

Later Reymonds group reported the synthesis of dendrimer 45 (Fig. 7.3) that catalyzes ester hydrolysis with a single catalytic histidine residue in the core [36]. According to the author, a pair of arginine residues in the first-generation branch assists substrate binding. 

Scheme 7.9 Proposed mechanism for the His-dendrimer-catalyzed ester hydrolysis (a) nucleophilic attack of histidine generates an acyl-dendrimer intermediate (b) general base catalyzed nucleophilic attack of a water molecule...

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