Role of pro-297 in the catalytic mechanism of sheep liver serine hydroxymethyltransferase.
Serine hydroxymethyltransferase belongs to the alpha 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 gamma-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.
Talwar
R
R
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Leelavathy
V
V
Krishna Rao
J V
JV
Appaji Rao
N
N
Savithri
H S
HS
D000818:Animals; D001483:Base Sequence; D001704:Biopolymers; D002384:Catalysis; D002942:Circular Dichroism; D017931:DNA Primers; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D011392:Proline / Q000378:metabolism*; D012756:Sheep; D013056:Spectrophotometry, Ultraviolet
D001704:Biopolymers; D017931:DNA Primers; D011392:Proline; D012696:Glycine Hydroxymethyltransferase