Role of His-224 in the anomalous pH dependence of human stromelysin-1.
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.
Holman
C M
CM
Inflammatory Diseases Unit, Roche Bioscience, Palo Alto, California 94304, USA.
Kan
C C
CC
Gehring
M R
MR
Van Wart
H E
HE
D005973:Glutamine / Q000235:genetics; D006466:Hemopexin / Q000737:chemistry; D006639:Histidine / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D019278:Matrix Metalloproteinase 3 / Q000737:chemistry / Q000235:genetics / Q000502:physiology*; D016297:Mutagenesis, Site-Directed; D010446:Peptide Fragments / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity
D010446:Peptide Fragments; D005973:Glutamine; D006639:Histidine; D006466:Hemopexin; D019278:Matrix Metalloproteinase 3