Catalysis by human leukocyte elastase: the proton inventory as a mechanistic probe

Abstract

Proton inventories (rate measurements in mixtures of H2O and D2O) were determined for the human leukocyte elastase catalyzed hydrolyses of thiobenzyl esters and p-nitroanilides of the peptides MeOSuc-Val, MeOSuc-Alan-Pro-Val (n = 0-2), and MeOSuc-Alan-Pro-Ala (n = 1 or 2). The dependencies of k2/Ks on mole fraction of solvent deuterium for the p-nitroanilides are "dome-shaped" and were fit to a model that incorporates the mechanistic features of generalized solvent reorganization when substrate binds to enzyme and partial rate limitation of k2/Ks by physical and chemical steps [Stein, R. L. (1985) J. Am. Chem. Soc. 107, 7768-7769]. The proton inventories for the deacylation of MeOSuc-Val-HLE and MeOSuc-Pro-Val-HLE are linear while those for the deacylation of MeOSuc-Ala-Pro-Val-HLE and MeOSuc-Ala-Ala-Pro-Val-HLE are "bowl-shaped" and could be fit to a quadratic dependence of rate on mole fraction of deuterium. These results are interpreted to suggest that the correct operation of the catalytic triad is dependent on substrate structure. Minimal substrates, which cannot interact with elastase at remote subsites, are hydrolyzed via a mechanism involving simple general-base catalysis by the active site histidine and transfer of a single proton in the rate-limiting transition state. In contrast, tri- and tetrapeptide substrates, which are able to interact at remote subsites, are hydrolyzed by a more complex mechanism of protolytic catalysis involving full functioning of the catalytic triad and transfer of two protons in the rate-limiting transition state. Finally, the proton inventories for the deacylation of MeOSuc-Ala-Pro-Ala-HLE and MeOSuc-Ala-Ala-Pro-Ala-HLE are dome-shaped and suggest that the chemical events of acyl-enzyme hydrolysis are only partially rate limiting for these reactions and that some other physical step is also partially rate limiting.

Structural origins of substrate discrimination in trypsin and chymotrypsin

“…Position 216 is Val in both pancreatic and human neutrophil elastases; in these enzymes this amino acid fulfills a dual role both by providing a hydrophobic platform for the P1 residue and by forming the main-chain hydrogen bonds at position P3 (Navia et al, 1989;Watson et al, 1970). Occupancy of the extended peptide binding site in both pancreatic and neutrophil elastases also increases the rate of acylation in preference to binding affinity or deacylation rate Stein et al, 1987). In the case of neutrophil elastase, it is also known that the interactions with the extended peptide binding site increase k2 for specific but not for nonspecific substrates (Stein et al, 1987).…”

“…Occupancy of the extended peptide binding site in both pancreatic and neutrophil elastases also increases the rate of acylation in preference to binding affinity or deacylation rate Stein et al, 1987). In the case of neutrophil elastase, it is also known that the interactions with the extended peptide binding site increase k2 for specific but not for nonspecific substrates (Stein et al, 1987). Therefore, we suggest that hydrogen bonds formed by the P3 residue Remington et al, 1988); PPE, CP = -133.8', Y = 176.7' (PDB 3EST; Meyer et al, 1988); and HNE, @ = -118.2', Y = 166.8' (PDB 1HNE; Navia et al, 1989).…”

Development of a sensitive peptidase assay: In search of cell associated proteases responsible for the cleavage of prepro TGFα

“…These interactions can be shown to influence catalysis such as is observed for the acid proteases (Moore et al, 1989), various metalloendoproteases (Powers and Harper, 1986), and elastases (Bode et al, 1989). Furthermore, the presence of a chromophoric leaving group may result in distorted specificity (Atlas and Berger, 1972) and proton inventory studies suggest that the catalytic mechanism for monomeric or dipeptide carrier substrates is distinct from that of extended peptides (Stein et al, 1987). While the usefulness of these synthetic substrates, which have well-defined chemical structures, in determining catalytic parameters or in the purification of enzyme(s) is well attested, their use to identify a n enzyme with tight substrate specificity may be inadequate.…”

Ionic Inhibitors of Human Leukocyte Elastase: Pyridinium and Phenyl Carboxylate Derivatives of 3-Alkyl-N-Hydroxysuccinimide

“…It would appear from the results shown in Table I that the overriding factor in affecting inhibitory activity is the nature of R,, a finding that is in accord with literature reports related to the inhibition of HLE by monomeric inhibitors. 14 The inhibitory activity of several of the inhibitors listed in Table I (1,3, and 8) was investigated using chymotrypsin and leukocyte cathepsin G. A 10-min incubation of each enzyme with a 200-fold excess of inhibitor resulted in near total inactivation (95-100%) of each proteinase. The proteinase-antiproteinase hypothesis concerning the pathogenesis of pulmonary emphysema proposes that neutrophils migrate through the alveolar interstitium and degranulate, releasing proteolytic enzymes, in particular elastase, into the interstitium.4J6J6 Elastase, if uninhibited, can bind to and degrade interstitial elastin.…”