Mechanistic studies on the human matrix metalloproteinase stromelysin

Abstract

To probe the mechanism of stromelysin (SLN)-catalyzed peptide hydrolysis, we determined the pH dependence of kc/Km and solvent deuterium isotope effects on kc and kc/Km. pH dependencies of kc/Km were determined for the SLN-catalyzed hydrolysis of three peptides: Arg-Pro-Lys-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2,Arg-Pro-Ala-Pro-Gln-Gln- Phe-Phe - Gly-Leu-NleNH2, and N-acetyl-Arg-Pro-Ala-Pro-Gln-Gln-Phe-Phe-Gly-Leu-Nle-NH2 (cleavage at Gln-Phe bond). The pH dependencies are all bell-shaped with shoulders that extend from pH 7.5 to 8.5. The existence of a shoulder indicates that the reaction mechanism involves at least two routes to products. These curves are governed by three proton ionizations with pKa values of 5.4, 6.1, and 9.5. The solvent isotope effect measurements provided the following values: D(kc/Km) = 0.80 +/- 0.05 and D(kc) = 1.58 +/- 0.05. That D(kc/Km) and D(kc) are different suggests that the rate-limiting transition states for the processes governed by kc/Km and kc cannot be the same. We use these results, together with analogy to thermolysin catalysis, to develop a mechanism for SLN catalysis.

pH stability of the stromelysin-1 catalytic domain and its mechanism of interaction with a glyoxal inhibitor

“…These free enzyme pK a values are very similar to the values of 5.4-5.8 and 6.0-6.3 obtained from plots of 1/K i versus pH with other inhibitors [32]. Plots of k cat /K M versus pH give similar free enzyme pK a values for pK 1 and pK 2 and they also give an additional pK a of 8.5-9.9 [32,33]. From the pH dependence of the K i values ( Fig.…”

“…As the positions of the signals did not change with pH, this decrease in intensity must be controlled by a slow exchange process. The pK a values of 7.94, 6.67 and 7.49 controlling the decrease in intensity of the signals at 12.9, 13.1 and 13.5 ppm respectively (Table 3), are significantly larger than pK 1 (5.4-5.8) and pK 2 (6.0-6.3) and smaller than pK 3 (8.5-9.9) determined from the pH dependence of k cat /K M [32,33]. It is therefore unlikely that these hydrogen bonded protons play a direct role in catalysis.…”

Catalytic Activities and Substrate Specificity of the Human Membrane Type 4 Matrix Metalloproteinase Catalytic Domain

“…The pH-activity profile for the MT4-MMPCD catalytic efficiency with FP2 resembles that reported for human stromelysin-1 (33,34) in that it is best fit to a model with three proton ionizations and two active enzyme forms that can lead to product. Since FP2 is not expected to ionize in the pH range studied, we conclude that these ionizations arise from groups on the free enzyme.…”

“…The observed k cat /K m data were fit to Equation 2 describing three proton ionizations (pK a values) and two limiting k cat /K m values as used previously with kinetic data for human stromelysin-1 (33).…”

Human matrix metalloproteinases: An ubiquitarian class of enzymes involved in several pathological processes

“…by both the Zn 2+ ion and a carbonyl group of a nearby peptide bond, leading to the cleavage of the scissile peptide bond when a second residue (usually a residue of His, which decreases its pK a ) donates a hydrogen atom to the nitrogen atom of the imido portion of the peptide bond (Harrison et al, 1992). This sequence of events is confirmed by the dramatic reduction of the MMP activity following the Glu residue mutation (Cha and Auld, 1997).…”