Effects of deletions at the carboxyl terminus of Zymomonas mobilis pyruvate decarboxylase on the kinetic properties and substrate specificity

Chang, AK, Nixon, PF and Duggleby, RG (2000) Effects of deletions at the carboxyl terminus of Zymomonas mobilis pyruvate decarboxylase on the kinetic properties and substrate specificity. Biochemistry, 39 31: 9430-9437. doi:10.1021/bi0002683


Author Chang, AK
Nixon, PF
Duggleby, RG
Title Effects of deletions at the carboxyl terminus of Zymomonas mobilis pyruvate decarboxylase on the kinetic properties and substrate specificity
Journal name Biochemistry   Check publisher's open access policy
ISSN 0006-2960
Publication date 2000-01-01
Sub-type Article (original research)
DOI 10.1021/bi0002683
Volume 39
Issue 31
Start page 9430
End page 9437
Total pages 8
Place of publication Washington
Publisher American Chemical Society
Collection year 2000
Language eng
Subject C1
270108 Enzymes
780105 Biological sciences
Abstract The three-dimensional structure of Zymomonas mobilis pyruvate decarboxylase shows that the carboxyl-terminal region of the protein occludes the active site. This observation is consistent with earlier suggestions that the active site is inaccessible to solvent during catalysis. However, the carboxy/terminal region must move aside to allow entry of the substrate, and again to permit the products to leave. Here we have examined the role of the carboxyl terminus by making 15 variants of the enzyme with serial deletions. The activity is largely unaffected by removal of up to seven residues but deletion of the next two, R561 and S560, results in a drastic loss of activity. Five of these deletion mutants were purified and fully characterized and showed progressive decreases in activity, in the ability to discriminate between pyruvate and larger substrates, and in cofactor affinity. Several substitution mutants at residues R561 and S560 were prepared, purified, and fully characterized. The results indicate important roles for the side-chain of R561 and the backbone atoms of S560. It is suggested that the carboxyl-terminal region of pyruvate decarboxylase is needed to lock in the cofactors and for the proper closure of the active site that is required for discrimination between substrates and for decarboxylation to occur.
Keyword Biochemistry & Molecular Biology
Site-directed Mutagenesis
Thiamin Diphosphate
Crystal-structure
Enzyme Action
Catalysis
Resolution
Mechanism
Cofactor
Magnesium
Linkage
Q-Index Code C1

Document type: Journal Article
Sub-type: Article (original research)
Collection: School of Biological Sciences Publications
 
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Created: Tue, 10 Jun 2008, 21:25:38 EST