Catalytic Cycling in -Phosphoglucomutase: A Kinetic and Structural Analysis
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- 作者：Guofeng Zhang ; Jianying Dai ; Liangbing Wang ; Debra Dunaway-Mariano ; Lee W. Tremblay ; and Karen N. Allen
- 刊名：Biochemistry
- 出版年：2005
- 出版时间：July 12, 2005
- 年：2005
- 卷：44
- 期：27
- 页码：9404 - 9416
- 全文大小：441K
- 年卷期：v.44,no.27(July 12, 2005)
- ISSN：1520-4995
文摘
Lactococcus lactis 
-phosphoglucomutase (-PGM) catalyzes the interconversion of -D-glucose1-phosphate (-G1P) and -D-glucose 6-phosphate (G6P), forming -D-glucose 1,6-(bis)phosphate (-G16P) as an intermediate. -PGM conserves the core domain catalytic scaffold of the phosphatase branchof the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as amutase rather than as a phosphatase. This work was carried out to identify the structural basis underlyingthis diversification of function. In this paper, we examine -PGM activation by the Mg2+ cofactor, -PGMactivation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First,the 1.90 Å resolution X-ray crystal structure of the Mg2+--PGM complex is examined in the context ofpreviously reported structures of the Mg2+-
-D-galactose-1-phosphate--PGM, Mg2+-phospho--PGM,and Mg2+--glucose-6-phosphate-1-phosphorane--PGM complexes to identify conformational changesthat occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmedby demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of theligands to Mg2+ in the different complexes shows that a single Mg2+ coordination site must alternativelyaccommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limitedinvolvement of the HAD family metal-binding loop in Mg2+ anchoring in -PGM is consistent with therelatively loose binding indicated by the large Km for Mg2+ activation (270 ± 20 
M) and with the retentionof activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of capand core domains in the different complexes indicated that interaction of cap domain Arg49 with the"nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation,as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analysesof the specificity of -PGM toward phosphoryl group donors and the specificity of phospho--PGM towardphosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity.Last, we present evidence that the autophosphorylation of -PGM by the substrate -G1P accounts forthe origin of phospho--PGM in the cell.
-phosphoglucomutase (-PGM) catalyzes the interconversion of -D-glucose1-phosphate (-G1P) and -D-glucose 6-phosphate (G6P), forming -D-glucose 1,6-(bis)phosphate (-G16P) as an intermediate. -PGM conserves the core domain catalytic scaffold of the phosphatase branchof the HAD (haloalkanoic acid dehalogenase) enzyme superfamily, yet it has evolved to function as amutase rather than as a phosphatase. This work was carried out to identify the structural basis underlyingthis diversification of function. In this paper, we examine -PGM activation by the Mg2+ cofactor, -PGMactivation by Asp8 phosphorylation, and the role of cap domain closure in substrate discrimination. First,the 1.90 Å resolution X-ray crystal structure of the Mg2+--PGM complex is examined in the context ofpreviously reported structures of the Mg2+--D-galactose-1-phosphate--PGM, Mg2+-phospho--PGM,and Mg2+--glucose-6-phosphate-1-phosphorane--PGM complexes to identify conformational changesthat occur during catalytic turnover. The essential role of Asp8 in nucleophilic catalysis was confirmedby demonstrating that the D8A and D8E mutants are devoid of catalytic activity. Comparison of theligands to Mg2+ in the different complexes shows that a single Mg2+ coordination site must alternativelyaccommodate water, phosphate, and the phosphorane intermediate during catalytic turnover. Limitedinvolvement of the HAD family metal-binding loop in Mg2+ anchoring in -PGM is consistent with therelatively loose binding indicated by the large Km for Mg2+ activation (270 ± 20 M) and with the retentionof activity found in the E169A/D170A double loop mutant. Comparison of the relative positions of capand core domains in the different complexes indicated that interaction of cap domain Arg49 with the"nontransferring" phosphoryl group of the substrate ligand might stabilize the cap-closed conformation,as required for active site desolvation and alignment of Asp10 for acid-base catalysis. Kinetic analysesof the specificity of -PGM toward phosphoryl group donors and the specificity of phospho--PGM towardphosphoryl group acceptors were carried out. The results support a substrate induced-fit mechanism of-PGM catalysis, which allows phosphomutase activity to dominate over the intrinsic phosphatase activity.Last, we present evidence that the autophosphorylation of -PGM by the substrate -G1P accounts forthe origin of phospho--PGM in the cell.