The role of lysine-256 in the structure and function of sheep liver recombinant serine hydroxymethyltransferase.
The active site lysine residue, K256, involved in Schiff's base linkage with pyridoxal-5'-phosphate (PLP) in sheep liver recombinant serine hydroxymethyltransferase (rSHMT) was changed to glutamine or arginine by site-directed mutagenesis. The purified K256Q and K256R SHMTs had less than 0.1% of catalytic activity with serine and H4folate as substrates compared to rSHMT. The mutant enzymes also failed to exhibit the characteristic visible absorbance spectrum (lambda(max) 425 nm) and did not produce the quinonoid intermediate (lambda(max) 495 nm) upon the addition of glycine and H4folate. The mutant enzymes were unable to catalyze aldol cleavage of beta-phenylserine and transamination of D-alanine. These results suggested that the mutation of the lysine had resulted in the inability of the enzyme to bind to the cofactor. Therefore, the K256Q SHMT was isolated as a dimer and the K256R SHMT as a mixture of dimers and tetramers which were converted to dimers slowly. On the other hand, rSHMT was stable as a tetramer for several months, further confirming the role of PLP in maintenance of oligomeric structure. The mutant enzymes also failed to exhibit the increased thermal stability upon the addition of serine, normally observed with rSHMT. The enhanced thermal stability has been attributed to a change in conformation of the enzyme from open to closed form leading to reaction specificity. The mutant enzymes were unable to undergo this conformational change probably because of the absence of bound cofactor.
Talwar
R
R
Department of Biochemistry, Indian Institute of Science, Bangalore.
Jagath
J R
JR
Datta
A
A
Prakash
V
V
Savithri
H S
HS
Rao
N A
NA
D000818:Animals; D001483:Base Sequence; D001665:Binding Sites / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D019281:Dimerization; D004795:Enzyme Stability; D012696:Glycine Hydroxymethyltransferase / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D066298:In Vitro Techniques; D008099:Liver / Q000201:enzymology; D008239:Lysine / Q000737:chemistry; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D012756:Sheep; D013053:Spectrophotometry
D017931:DNA Primers; D011994:Recombinant Proteins; D012696:Glycine Hydroxymethyltransferase; D008239:Lysine