Substrate positioning by His92 is important in catalysis by purple acid phosphatase

被引:29
作者
Funhoff, EG
Wang, WL
Andersson, G
Averill, BA
机构
[1] Univ Toledo, Dept Chem, Toledo, OH 43606 USA
[2] Univ Amsterdam, Swammerdam Inst Life Sci, NL-1012 WX Amsterdam, Netherlands
[3] Karolinska Inst, Huddinge Univ Hosp, Div Pathol, S-10401 Stockholm, Sweden
[4] ETH Honggerberg, HPT, Inst Biotechnol, CH-8093 Zurich, Switzerland
关键词
kinetics; mechanism; mutagenesis; purple acid phosphatase; spectroscopy;
D O I
10.1111/j.1742-4658.2005.04686.x
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Proteolysis of single polypeptide mammalian purple acid phosphatases (PAPs) results in the loss of an interaction between the loop residue Asp146 and the active site residues Asn91 and/or His92. While Asn91 is a ligand to the divalent metal of the mixed-valent di-iron center, the role of His92 in the catalytic mechanism is unknown. Site-directed mutagenesis of His92 was performed to examine the role of this residue in single polypeptide PAP. Conversion of His92 into Ala, which eliminates polar interactions of this residue with the active site, resulted in a 10-fold decrease in catalytic activity at the optimal pH. Conversely, conversion of this residue into Asn, which cannot function as either a proton donor or acceptor, but can provide hydrogen-bonding interactions, resulted in a three-fold increase in activity at the optimal pH. Both mutant enzymes had more acidic pH optima, with pK(es,1) values consistent with the involvement of an iron(III) hydroxide unit or a hydroxide in the second coordination sphere in catalysis. These results, together with EPR data, support a role of His92 in positioning either the nucleophile or the substrate, rather than directly in acid or base catalysis. The existence of an extensive hydrogen-bonding network that could fine-tune the position of His92 is consistent with this proposal.
引用
收藏
页码:2968 / 2977
页数:10
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