Structural Analyses on Intermediates in Serine Protease Catalysis

Abstract

Although the subject of many studies, detailed structural information on aspects of the catalytic cycle of serine proteases is lacking. Crystallographic analyses were performed in which an acyl-enzyme complex, formed from elastase and a peptide, was reacted with a series of nucleophilic dipeptides. Multiple analyses led to electron density maps consistent with the formation of a tetrahedral species. In certain cases, apparent peptide bond formation at the active site was observed, and the electron density maps suggested production of a cis-amide rather than a trans-amide. Evidence for a cis-amide configuration was also observed in the noncovalent complex between elastase and an ␣ 1 -antitrypsin-derived tetrapeptide. Although there are caveats on the relevance of the crystallographic data to solution catalysis, the results enable detailed proposals for the pathway of the acylation step to be made. At least in some cases, it is proposed that the alcohol of Ser-195 may preferentially attack the carbonyl of the cis-amide form of the substrate, in a stereoelectronically favored manner, to give a tetrahedral oxyanion intermediate, which undergoes N-inversion and/or C-N bond rotation to enable protonation of the leaving group nitrogen. The mechanistic proposals may have consequences for protease inhibition, in particular for the design of high energy intermediate analogues.Following from their role in the development of mechanistic and structural enzymology, serine proteases, and more generally enzymes catalyzing hydrolysis or related reactions proceeding via an acyl-enzyme complex, have been shown to be of importance in human diseases. Serine proteases, including thrombin, factor X, and elastase, are targets for medicinal chemistry (1, 2). One important class of inhibitors is designed to mimic a tetrahedral intermediate, but until recently there has been little three-dimensional structural information on the early intermediates in catalysis.In outline, the accepted mechanism for serine proteases involves nucleophilic attack by the alcohol of Ser-195 onto the amide carbonyl of the peptide substrate to form a first tetrahedral intermediate. This intermediate collapses to form an ester, or acyl-enzyme complex, and the COOH-terminal product fragment then leaves the active site. Hydrolysis of the Ser-195-linked ester bond then occurs via a second tetrahedral intermediate, to form the NH 2 -terminal product fragment and return the active site to its resting state. Much evidence has accumulated in support of this general mechanism, including the role of His-57 in enabling acid/base catalysis, and it has been extended to other types of hydrolysis reaction, including esterases. Although there has been extensive structural work on covalently bound inhibitors and some on substrate analogues (3), there is relatively little direct crystallographic evidence for the key intermediates, and questions remain as to the precise stereoelectronic pathway of the reaction, including the timing and nature of the requisite proton transfers (4)...

A critical electrostatic interaction mediates inhibitor recognition by human asparagine synthetase

“…For example, only a single enantiomer of a given sulfoximine derivative exhibits potent inhibitor activity in experiments where diastereoisomeric and/or enantiomeric mixtures of sulfoximines have been separated. 37,38 Additional support for this assumption is provided by experimental 39,40 and computational studies 41 showing that hydrogen bonding interactions stabilize a single epimer of the tetrahedral intermediate formed when nucleophiles react with activated carbonyl groups. Assuming that only one of the four diastereoisomers of 3 is active, then the K Ã I value demonstrates that the sulfamide derivative is three orders of magnitude less effective an inhibitor of human ASNS than the adenylated sulfoximine 1.…”

The Road to Avibactam: The First Clinically Useful Non-β-Lactam Working Somewhat Like a β-Lactam

“…In this regard, the Tipper-Strominger hypothesis [67], in other words, that β-lactam antibacterials mimic the conformation of the D-Ala-D-Ala component of the PBP bacterial cell wall substrate, may be relevant [68], in that in order to get to the stage where reaction to the acylenzyme complex can occur the β-lactam antibacterial must efficiently compete with the natural substrate for binding [69]. Following from biophysical evidence that some nucleophilic serine enzymes may bind their substrates in a high-energy conformation which strains the amide bond [70][71][72], a refinement of the Tipper-Strominger hypothesis suggests that (some) β-lactam antibacterials may mimic a strained conformation of the D-Ala-D-Ala entity (Figure 2) [73][74][75]. Thus, (at least some) β-lactam antibacterials may be highly optimized to bind both noncovalently and covalently; following noncovalent binding, they undergo covalent reaction to form an initial acyl-enzyme complex which at least, in some cases, can undergo either covalent fragmentation [52,53,76] or conformational rearrangement [23,52,[55][56][57]59,62,77] to form (a) more stable complexes.…”

Restricting the ψ Torsion Angle Has Stereoelectronic Consequences on a Scissile Bond: An Electronic Structure Analysis

“…A few reports suggest the key to activation is rotation around the ω torsion angle in the form of cis – trans isomerization. 20,21 Other studies argue that scissile bond distortion depends on ψ , as imidic bond twisting and carbonyl pyramidalization occur over a range of ψ angles (i.e., when ψ is close to ±30°, ±90°, and ±150°). 22–26 …”