THE ROLE OF LYSINE-256 IN THE STRUCTURE AND FUNCTION OF SHEEP LIVER RECOMBINANT SERINE HYDROXYMETHYLTRANSFERASE

The active site lysine residue, K256, involved in Schiffs base linkage with pyridoxal-5'-phosphate (PEP) in sheep liver recombinant serine h ydroxymethyltransferase (rSHMT) was changed to glutamine or arginine b y site-directed mutagenesis. The purified K256Q and K256R SHMTs had le ss than 0.1% of catalytic activity with serine and H(4)folate as subst rates compared to rSHMT. The mutant enzymes also failed to exhibit the characteristic visible absorbance spectrum (lambda(max) 425 nm) and d id not produce the quinonoid intermediate (lambda(max) 495 nm) upon th e addition of glycine and H(4)folate. The mutant enzymes were unable t o 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. There fore, the K256Q SHMT was isolated as a dimer and the K256R SHMT as a m ixture 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 struct ure. The mutant enzymes also failed to exhibit the increased thermal s tability upon the addition of serine, normally observed with rSHMT. Th e enhanced thermal stability has been attributed to a change in confor mation of the enzyme from open to closed form leading to reaction spec ificity. The mutant enzymes were unable to undergo this conformational change probably because of the absence of bound cofactor.