Analysis of an enzyme-substrate complex by x-ray crystallography and transferred nuclear overhauser enhancement measurements: porcine pancreatic elastase and a hexapeptide

Abstract

The hexapeptide substrate Thr-Pro-nVal-NMeLeu-Tyr-Thr reacts with porcine pancreatic elastase sufficiently slowly that accelerated crystallographic data collection procedures and two-dimensional transferred nuclear Overhauser enhancement measurements could be used to study the geometry of binding. Both studies report a time-averaged population of the Michaelis complex state, prior to proteolysis. This result provides an important data point along the reaction coordinate pathway for serine proteases. Crystallographic data to 1.80-A resolution were used in the structure analysis with refinement to an R-factor of 0.19.W ith the exception of inhibited complexes, most enzymatic reactions are opaque to investigation of structure-functional relationships in and around the active site. A number of details about the reaction mechanism of the serine proteases therefore remain poorly understood. In this paper we describe two complementary experiments designed to probe structural details of a kinetically retarded reaction involving porcine pancreatic elastase (PPE).lOn the basis of molecular orbital considerations proposed by Deslongchamps (1975), Bizzozero and Zweifel (1975) showed that a-chymotrypsin does not cleave a peptide (or ester) bond when the "leaving-group" N (or C) atom is alkylated (Pro or sarcosine). Bizzozero and Dutler (1981) extended this study to suggest that a stereochemical inversion is the committing step in proteolysis; a methylated peptidebond N atom is thereby restrained from rapid inversion. The slow turnover of the reaction described here supports these hypotheses and provides a means to view structural interactions within a functioning enzyme. Our first attempt to test this hypothesis with PPE using as a substrate Pro-Ala-Pro-Tyr led to backward, nonproductive binding (Clore et al., 1986a). We therefore chose to study a hexapeptide, Thr-Pro-nVal-NMeLeu-Tyr-Thr, designed to bind unambiguously to porcine pancreatic elastase. As before (Clore et al., 1986a), a dual-pronged attack, using both high-resolution crystallography and N M R spectroscopy involving the use of TRNOE measurements (Clore & Gronenborn, 1982, 1983, was initiated. Because of the lability of the complex, the crystallographic data collection was limited by a regimen chosen to ensure adequate ligand binding: 24 h of soaking and 48 h of data collection. Such a regimen places a severe strain on conventional crystallographic measurements, which traditionally can require weeks to months to complete. Similarly, the N M R measurements were limited to 24 h of data collection per sample. EXPERIMENTAL PROCEDURESSamples. PPE was purchased from Serva (20931) and used without further purification. The hexapeptide Thr-PronVal-NMeLeu-Tyr-Thr was synthesized by Bachem (Switz- 0006-2960/88/0427-0725$01 .50/0 erland) and was >99.5% pure as judged by HPLC. Crystallography. Crystals of PPE were grown from a 0.1 M phosphate buffer (pH 5.0) (Meyer et al., 1986) and harvested with a 0.1 M sulfate plus 0.1 M phosphate (pH 5.0) buffer. A crystal (0...

Human Cytomegalovirus Protease Complexes Its Substrate Recognition Sequences in an Extended Peptide Conformation,

“…NMR studies involving full processing site sequences would undoubtedly represent the most direct approach to obtaining information concerning substrate binding (30). Although the proton NMR spectrum of a P9-P8′ R-site substrate mimic R-R′ could be obtained in the presence of HCMV protease, which indeed exhibited changes in certain proton resonances as compared to that of the free peptide, the rapid enzymatic cleavage to product fragments and the emergence of new signals precluded any detailed NMR studies of the bound and intact processing site sequences.…”

Internal water molecules and H‐bonding in biological macromolecules: A review of structural features with functional implications

“…fig. 4, Meyer & Bode, 1987) for the superposition of PPE and HLE). Moreover, enzymes of this family exhibit little structural variation upon substrate binding, e.g., the various structures of bound and free trypsin have RMS differences of ca.…”

“…S4 to S3') binding site: For trypsin, PPE, HLE, etc. upon forming the Michaelis complex with a peptide-like substrate occupying both S and S' regions of the extended binding site (notation of Schechter & Berger [196719), bulk water is effectively excluded from the active site (Meyer et al, 1988a;Dauter et al, 1991), with one possible exception:…”

Citrate-Linked Keto- and Aldo-Hexose Monosaccharide Cellulose Conjugates Demonstrate Selective Human Neutrophil Elastase-Lowering Activity in Cotton Dressings

“…There is precedent for this type of binding interaction with elastase in the inhibitor ursolic acid which also possesses a carboxyl negative charge that interacts with Arg-217 [50]. In addition the side chain hydroxyls of the threonine residues in a well characterized crystal structure [51] of porcine pancreatic elastase with the threonine hexapeptide serve as an indication that hydroxyl functionalities as those found in the anomeric pyranose and furanose rings are accommodated in the active site. This proposed mechanism of the monosaccharide conjugate’s enzyme active site binding is analogous to elastase inhibitor binding found in certain haloalkylketone- and aldehyde-based inhibitors, as previously reported [52].…”