The importance of aspartate 327 for catalysis and zinc binding in Escherichia coli alkaline phosphatase.

X Xu,E.R Kantrowitz

doi:10.1016/s0021-9258(18)41992-8

Abstract

In order to investigate the function of Asp-327, a bidentate ligand of one of the zinc atoms in Escherichia coli alkaline phosphatase, and the importance of this zinc atom in catalysis, site-specific mutagenesis was used to convert Asp-327 to either asparagine or alanine. The 10(7)-fold decrease in the kcat/Km ratio observed for the Asp-327----Ala enzyme compared to the wild-type enzyme indicates that the side chain of Asp-327 is important for zinc binding at the M1 site. However, only one of the two carboxyl oxygens of Asp-327 is essential for zinc binding, since the Asp-327----Asn enzyme shows approximately the same hydrolysis activity as the wild-type enzyme. The fact that the enzymatic activity of this mutant enzyme shows a dependence on zinc concentration suggests that the other carboxyl oxygen or the negative charge on the side chain of Asp-327 is important in binding of the zinc at the M1 site. However, the zinc hydroxyl must still be appropriately positioned to attack the phosphoserine in the Asp-327----Asn enzyme; therefore, the negative charge and at least one carboxyl oxygen of the side chain are not directly involved in positioning or deprotonating the zinc hydroxyl. 31P NMR studies indicate that the Asp-327----Asn enzyme exhibits transphosphorylation activity at both pH 8.0 and pH 10.0, but at a reduced level compared to the wild-type enzyme. The biphasic production of 2,4-dinitrophenylate in the pre-steady-state kinetics of the mutant enzymes at pH 5.5 suggests that the breaking of the phosphoenzyme covalent complex is rate-limiting for both mutant enzymes. These results suggest that the main function of the zinc atom at the M1 site in catalysis involves decomposition of the phosphoenzyme covalent complex and that it may be important in helping to stabilize the alcohol leaving group.

Highlights

Carboxyl oxygen or the negative charge on the side Theimportance of zinc inbothcatalysisandstructural chain of Asp-327 is important in bindingof the zinc at stabilization of alkalinephosphatasehas longbeen recogthe M1 site
Studies of the reactivation of the apoenchain arenot directly involved in positionoinrgdepro- zyme by metal ions have shown that at least two zinc atoms tonating the zinc hydroxyl. 31PNMR studies indicate per mol of enzyme dimer are required to restore enzymatic that the Asp-327 + Asn enzyme exhibits transphos- activity (Plocke andVallee, 1962; Simpson and Vallee, 1968; phorylation activity at both pH 8.0 and pH 10.0, but Harris andColeman, 1968; Anderson et al, 1975)
These results suggest that themain function of the zinc atom at the M1 site in catalysis involves decomposition of the phosphoenzyme covalent complex and that mit ay be important inhelping to stabilizethe alcohol leaving group

Summary

Introduction

Carboxyl oxygen or the negative charge on the side Theimportance of zinc inbothcatalysisandstructural chain of Asp-327 is important in bindingof the zinc at stabilization of alkalinephosphatasehas longbeen recogthe M1 site. Consistent with the fact thaztinc binds to thewild-type enzyme tightly (KO< lo-' M ) a t both the M1 and M2 sites (Coleman et al, 1983), the hydrolysis activity of the wild-type enzyme measured in a buffer system that has no phosphate acceptor (0.01 M Tris) shows no sensitivity to the concentratioonf zinc in the rangebetween and M (Fig. 2).

Results

Conclusion