Publication Date:
2012-04-27
Description:
Human matrix metalloproteinase 7 (MMP-7) activity exhibits broad bell-shaped pH profile with the acidic and alkaline p K a (p K e1 and p K e2 ) values of about 4 and 10. The ionizable group for p K e2 was assigned to Lys or Arg by thermodynamic analysis; however, no such residues are present in the active site. Hence, based on the crystal structure, we hypothesized that a water molecule bound to the main-chain nitrogen of Ala162 (W1) or the main-chain carbonyl oxygen of Pro217 (W2) is a candidate for the ionizable group for p K e2 [Takeharu, H. et al. (2011) Biochim. Biophys. Acta 1814, 1940–1946]. In this study, we inspected this hypothesis. In the hydrolysis of (7-methoxycoumarin-4-yl)acetyl- L -Pro- L -Leu–Gly- L -Leu-[ N 3 -(2,4-dinitrophenyl)- L -2,3-diaminopropionyl]- L -Ala- L -Arg-NH 2 , all 19 variants, in which one of all Lys and Arg residues was replaced by Ala, retained activity, indicating that neither Lys nor Arg is the ionizable group. p K e2 values of A162S, A162V and A162G were 9.6 ± 0.1, 9.5 ± 0.1 and 10.4 ± 0.2, respectively, different from that of wild-type MMP-7 (WT) (9.9 ± 0.1) by 0.3–0.5 pH unit, and those of P217S, P217V and P217G were 10.1 ± 0.1, 9.8 ± 0.1 and 9.7 ± 0.1, respectively, different from that of WT by 0.1–0.2 pH unit. These results suggest a possibility of W1 or W2 as the ionizable group for p K e2 .
Print ISSN:
0021-924X
Electronic ISSN:
1756-2651
Topics:
Biology
,
Chemistry and Pharmacology
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