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

详细信息    查看全文

• 作者：Christopher M. Holman ; Chen-Chen Kan ; Michael R. Gehring ; and Harold E. Van Wart
• 刊名：Biochemistry
• 出版年：1999
• 出版时间：January 12, 1999
• 年：1999
• 卷：38
• 期：2
• 页码：677 - 681
• 全文大小：49K
• 年卷期：v.38,no.2(January 12, 1999)
• ISSN：1520-4995

文摘

A plot of the pH dependence of k_cat/K_M for human stromelysin-1 (HS) exhibits a narrow rangeof maximal activity extending from pH 5.75 to 6.25 and a broad shoulder in the pH range of 7.5-8.5. Incontrast, 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 dependenceof 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 testthis hypothesis, the H224Q mutant of the short form (lacking the C-terminal hemopexin-like domain) ofHS (sHS) has been prepared and studied. The pH profile of H224Q sHS is bell-shaped and similar tothose reported for other MMPs. Although H224Q and wild-type sHS possess similar activities at pH <6,the k_cat/K_M of H224Q sHS is more than 5-fold greater than that of the wild-type enzyme at pH >7. Thesedata strongly suggest that the deprotonation of His-224 attenuates the activity of HS, thereby accountingfor its low pH optimum and the characteristic shoulder in its pH profile. This attenuation of activityappears to be predominantly a K_M effect, reflecting a decrease in the affinity of the enzyme for the peptidesubstrate.