Binding to thermolysin of phenolate-containing inhibitors necessitates a revised mechanism of catalysis

Abstract

Competitive inhibition as a function of pH for the metalloendoprotease thermolysin by derivatives of L-alpha-(2-hydroxyphenyl)benzenepropanoyl-L- tryptophanylglycylglycine exhibits a diagnostic bell shape. Binding is maximal between two pKa values: on the acidic limb the apparent Ki value is regulated by an unchanging enzymic ionization (pKa 5.3) which is also seen in the substrate-hydrolysis kinetics (kcat/Km), whereas the alkaline limb for inhibition varies and depends specifically on the pKa of the phenolic group in the inhibitor. Although it should be the phenolate form of the inhibitor that co-ordinates more efficiently to the active-site Zn2+, the apparent Ki shifts from pH-independent at pH values immediately below the inhibitor's pKa to progressively weaker binding at higher pH. This is explained by an anomalous acidity for the exchangeable solvent molecule that is attached to enzymic Zn2+ in the absence of substrate or inhibitor. Since OH- cannot be displaced from the enzyme as readily as H2O, a compensating pKa of 5.3 possessed by Zn(2+)-bound water rationalizes the binding characteristics, yielding the level pH profile exhibited at intermediate pH values. Recognition of the implicit heightened Lewis acidity of the metal ion in thermolysin leads to a revision of the mechanism of catalysis. The substrate amide bond becomes activated for hydrolysis by carbonyl-group co-ordination to the especially acidic Zn2+ ion (completely displacing the H2O/OH- species otherwise bound). The imidazole group of enzymic residue His-231, also discerned in the pH profile for kcat/Km from its pKa of 8, provides general-base assistance for hydration of the activated scissile linkage in the first committed step of catalysis. Additional evidence from inhibition patterns shows how substrate-binding energy may be employed in this scheme to promote hydrolysis of peptides by thermolysin.

Mutational analysis of the proteolytic domain of pregnancy-associated plasma protein-A (PAPP-A): classification as a metzincin

“…Although it is generally accepted, and supported by numerous studies on similar enzymes [31][32][33][34], the concept that the conserved Glu in enzymes with either of the two zinc-binding motifs functions as a general base catalyst in substrate hydrolysis has been contradicted [35][36][37]. Thus residues outside the zinc-binding motif have been proposed to play the role of the general base.…”

Effects of Site-Directed Mutagenesis of the Loop Residue of the N-Terminal Domain Gly117 of Thermolysin on Its Catalytic Activity

“…9) It consists of a -rich N-terminal domain and an -helical C-terminal domain. [10][11][12] The active site of thermolysin is composed of one zinc ion and five polypeptide regions: an N-terminal strand (Asn112-Trp115), -helix 1 (Val139-Thr149), C-terminal loop 1 (Asp150-Gly162), -helix 2 (Ala163-Val176), and C-terminal loop 2 (Gln225-Ser234).…”

The Effects of Modifying the Surface Charge on the Catalytic Activity of a Thermolysin-like Protease

“…From x-ray structures of thermolysin⅐inhibitor complexes (5, 9 -13), the active site residues have been identified and a mechanism has been proposed (13)(14)(15). Recently, an alternative mechanism has been proposed that has gained some support (16,17). In both proposed mechanisms residues Glu-143, His-231, Tyr-157, and a Zn 2ϩ bound water play important roles during catalysis.…”