Squash glycerol-3-phosphate (1)-acyltransferase. Alteration of substrate selectivity and identification of arginine and lysine residues important in catalytic activity
| Autor: | Antoni R, Slabas, Johan T M, Kroon, Ted P, Scheirer, John S, Gilroy, Matthew, Hayman, David W, Rice, Andy P, Turnbull, John B, Rafferty, Tony, Fawcett, William J, Simon |
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| Rok vydání: | 2002 |
| Předmět: |
Models
Molecular Binding Sites DNA Complementary Time Factors Sequence Homology Amino Acid Lysine Phenylalanine Molecular Sequence Data Sequence Analysis DNA Arginine Crystallography X-Ray Recombinant Proteins Substrate Specificity Kinetics Cucurbita Leucine Catalytic Domain Glycerol-3-Phosphate O-Acyltransferase Mutation Mutagenesis Site-Directed Histidine Amino Acid Sequence Protein Binding |
| Zdroj: | The Journal of biological chemistry. 277(46) |
| ISSN: | 0021-9258 |
| Popis: | Glycerol-3-phosphate 1-acyltransferase is a soluble chloroplast enzyme involved in glycerol-lipid biosynthesis associated with chilling resistance in plants (). Resistance is associated with higher selectivity for unsaturated acyl substrates over saturated ones. In vitro substrate selectivity assays performed under physiologically relevant conditions have been established that discriminate between selective and non-selective forms of the enzyme. A mutation, L261F, in the squash protein converts it from a non-selective enzyme into a selective one. The mutation lies within 10 A of the predicted acyl binding site and results in a higher K(m) for 16:0 acyl carrier protein (ACP). Site-directed mutagenesis was used to determine the importance of four residues, Arg(235), Arg(237), Lys(193), and His(194), implicated to be involved in binding of the phosphate group of glycerol 3-phosphate to the enzyme. All the proteins were highly homologous in structure to the wild type enzyme. Mutations in Arg(235), Arg(237), and Lys(193) resulted in inactive enzyme, while His(194) had reduced catalytic activity. The mutant proteins retained the ability to bind stoichiometric quantities of acyl-ACPs supporting the potential role of these residues in glycerol 3-phosphate binding. |
| Databáze: | OpenAIRE |
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