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Escherichia coli alkaline phosphatase: X‐ray structural studies of a mutant enzyme (His‐412 → Asn) at one of the catalytically important zinc binding sites
Author(s) -
Ma Lan,
Tibbitts Thomas T.,
Kantrowitz Evan R.
Publication year - 1995
Publication title -
protein science
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 3.353
H-Index - 175
eISSN - 1469-896X
pISSN - 0961-8368
DOI - 10.1002/pro.5560040807
Subject(s) - chemistry , zinc , enzyme , active site , alkaline phosphatase , escherichia coli , binding site , tris , hydrolase , stereochemistry , phosphatase , mutant , site directed mutagenesis , substrate (aquarium) , crystallography , biochemistry , biology , organic chemistry , ecology , gene
The X‐ray structure of a mutant version of Escherichia coli alkaline phosphatase (H412N) in which His‐412 was replaced by Asn has been determined at both low (‐Zn) and high (+Zn) concentrations of zinc. In the wild‐type structure, His‐412 is a direct ligand to one of the two catalytically critical zinc atoms (Zn,) in the active site. Characterization of the H412N enzyme in solution revealed that the mutant enzyme required high concentrations of zinc for maximal activity and for high substrate and phosphate affinity (Ma L, Kantrowitz ER, 1994, J Biol Chem 269 :31614–31619). The H412N enzyme was also inhibited by Tris, in contrast to the wild‐type enzyme, which is activated more than twofold by 1 M Tris. To understand these kinetic properties at the molecular level, the structure of the H412N(+Zn) enzyme was refined to an R ‐factor of 0.174 at 2.2 Å resolution, and the structure of the H412N(‐Zn) enzyme was refined to an R ‐factor of 0.166 at a resolution of 2.6 Å. Both indicated that the Asn residue substituted for His‐412 did not coordinate well to Zn 1 . In the H412N(‐Zn) structure, the Zn, site had very low occupancy and the phosphate was shifted by 1.8 Å from its position in the wild‐type structure. The Mg binding site was also affected by the substitution of Asn for His‐412. Both structures of the H412N enzyme also revealed a surface‐accessible cavity near the Zn, site that may serve as a binding site for Tris. The binding of Tris at the Zn, site may block Zn 2+ binding at this site, which would account for the reduced affinity of the H412N enzyme for substrate as well as its reduced catalytic efficiency in the presence of Tris. Thus, the replacement of His‐412 with Asn affects all three metal binding sites, suggesting that the relative positions of the metals bound at the active site of E. coli alkaline phosphatase are strongly linked and critical for efficient catalysis.