Role of His-224 in the Anomalous pH Dependence of Human Stromelysin-1

Abstract

A plot of the pH dependence of kcat/KM for human stromelysin-1 (HS) exhibits a narrow range of maximal activity extending from pH 5.75 to 6.25 and a broad shoulder in the pH range of 7.5-8.5. In contrast, the pH profiles that have been reported for other members of the matrix metalloproteinase (MMP) family are bell-shaped and exhibit neutral pH optima. We hypothesized that the anomalous pH dependence of HS reflects the ionization of His-224, a residue located in a flexible loop that contributes to the S1' binding pocket of the enzyme. HS is the only known MMP that has a histidine in this position. To test this hypothesis, the H224Q mutant of the short form (lacking the C-terminal hemopexin-like domain) of HS (sHS) has been prepared and studied. The pH profile of H224Q sHS is bell-shaped and similar to those reported for other MMPs. Although H224Q and wild-type sHS possess similar activities at pH <6, the kcat/KM of H224Q sHS is more than 5-fold greater than that of the wild-type enzyme at pH >7. These data strongly suggest that the deprotonation of His-224 attenuates the activity of HS, thereby accounting for its low pH optimum and the characteristic shoulder in its pH profile. This attenuation of activity appears to be predominantly a KM effect, reflecting a decrease in the affinity of the enzyme for the peptide substrate.

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.…”

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

“…This sequence of events is confirmed by the dramatic reduction of the MMP activity following the Glu residue mutation (Cha and Auld, 1997). A consequence of this protonation/deprotonation sequence of events is a complex pH-dependence of the enzymatic mechanism, which has been investigated in several MMPs Stack and Gray, 1990); in particular, site-directed mutagenesis has allowed to identify in MMP-3 the crucial role of His 224 as a proton donor (Holman et al, 1999;Johnson et al, 2000).…”

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

“…For most metalloproteinases the pH dependence of k cat /K M is bell shaped being dependant on three protonic states (EH 2 , EH and E) with the EH state being the catalytically active form (Scheme 3A) whose formation is governed by pK 1 and whose breakdown is governed by pK 2 [38]. However, for SCD the pH dependance of k cat /K M is more complex [32,33,38] being dependent on 4 protonic states (EH 3 , EH 2 , EH and E) with two catalytically active states (EH 2 and EH) whose formation and breakdown are controlled by the free enzyme pK a s pK 1 , pK 2 and pK 3 (Scheme 3B).…”

“…However, for SCD the pH dependance of k cat /K M is more complex [32,33,38] being dependent on 4 protonic states (EH 3 , EH 2 , EH and E) with two catalytically active states (EH 2 and EH) whose formation and breakdown are controlled by the free enzyme pK a s pK 1 , pK 2 and pK 3 (Scheme 3B). pK 1 , pK 2 and pK 3 give free enzyme pK a values of 5.2-5.8, 6.0-6.3 and 8.7-9.9 respectively [32,33,38]. pK 1 has been assigned to a Glu-202-zinc-water complex [32,33,38] and pK 3 is assigned to Tyr-223 [38].…”