Analysis of the Substrate Specificity Loop of the HAD Superfamily Cap Domain
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- 作者：Sushmita D. Lahiri ; Guofeng Zhang ; Jianying Dai ; Debra Dunaway-Mariano ; and Karen N. Allen
- 刊名：Biochemistry
- 出版年：2004
- 出版时间：March 16, 2004
- 年：2004
- 卷：43
- 期：10
- 页码：2812 - 2820
- 全文大小：664K
- 年卷期：v.43,no.10(March 16, 2004)
- ISSN：1520-4995
文摘
The haloacid dehalogenase (HAD) superfamily includes a variety of enzymes that catalyzethe cleavage of substrate C-Cl, P-C, and P-OP bonds via nucleophilic substitution pathways. All memberspossess the 
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core domain, and many also possess a small cap domain. The active site of the coredomain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate andcofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domainis responsible for the diversification of chemistry within the family. A tight -turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked byresidues whose side chains contribute to the catalytic site formed at the domain-domain interface. Todefine the role of the conserved Gly in the structure and function of the cap domain loop of the HADsuperfamily members phosphonoacetaldehyde hydrolase and -phosphoglucomutase, the Gly was mutatedto Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impactof Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had adramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the SchiffBase forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fillits place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Glyis critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificityloop can be identified within the amino acid sequence of HAD family members.
/ core domain, and many also possess a small cap domain. The active site of the coredomain is formed by four loops (corresponding to sequence motifs 1-4), which position substrate andcofactor-binding residues as well as the catalytic groups that mediate the "core" chemistry. The cap domainis responsible for the diversification of chemistry within the family. A tight -turn in the helix-loop-helix motif of the cap domain contains a stringently conserved Gly (within sequence motif 5), flanked byresidues whose side chains contribute to the catalytic site formed at the domain-domain interface. Todefine the role of the conserved Gly in the structure and function of the cap domain loop of the HADsuperfamily members phosphonoacetaldehyde hydrolase and -phosphoglucomutase, the Gly was mutatedto Pro, Val, or Ala. The catalytic activity was severely reduced in each mutant. To examine the impactof Gly substitution on loop 5 conformation, the X-ray crystal structure of the Gly50Pro phosphonoacetaldehyde hydrolase mutant was determined. The altered backbone conformation at position 50 had adramatic effect on the spatial disposition of the side chains of neighboring residues. Lys53, the SchiffBase forming lysine, had rotated out of the catalytic site and the side chain of Leu52 had moved to fillits place. On the basis of these studies, it was concluded that the flexibility afforded by the conserved Glyis critical to the function of loop 5 and that it is a marker by which the cap domain substrate specificityloop can be identified within the amino acid sequence of HAD family members.