Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase

Talwar, Rashmi ; Leelavathy, Vijayapandian ; Krishna Rao, Jala V. ; Appaji Rao, Naropantul ; Savithri, H. S. (2000) Role of Pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase Biochemical Journal, 350 . pp. 849-853. ISSN 0264-6021

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Serine hydroxymethyltransferase belongs to the α class of pyridoxal-5′-phosphate enzymes along with aspartate aminotransferase. Recent reports on the three-dimensional structure of human liver cytosolic serine hydroxymethyltransferase had suggested a high degree of similarity between the active-site geometries of the two enzymes. A comparison of the sequences of serine hydroxymethyltransferases revealed the presence of several highly conserved residues, including Pro-297. This residue is equivalent to residue Arg-292 of aspartate aminotransferase, which binds the γ-carboxy group of aspartate. In an attempt to change the reaction specificity of the hydroxymethyltransferase to that of an aminotransferase and to assign a possible reason for the conserved nature of Pro-297, it was mutated to Arg. The mutation decreased the hydroxymethyltransferase activity significantly (by 85-90%) and abolished the ability to catalyse alternative reactions, without alteration in the oligomeric structure, pyridoxal 5′-phosphate content or substrate binding. However, the concentration of the quinonoid intermediate and the extent of proton exchange was decreased considerably (by approx. 85%) corresponding to the decrease in catalytic activity. Interestingly, mutant Pro-297 Arg was unable to perform the transamination reaction with L-aspartate. All these results suggest that although Pro-297 is indirectly involved in catalysis, it might not have any role in imparting substrate specificity, unlike the similarly positioned Arg-292 in aspartate aminotransferase.

Item Type:Article
Source:Copyright of this article belongs to Portland Press Limited.
Keywords:Proton Abstraction; Pyridoxal-5′-phosphate; Reaction Specificity; Site-directed Mutagenesis
ID Code:21140
Deposited On:20 Nov 2010 09:04
Last Modified:17 May 2016 05:21

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