Purification and regulatory properties of mung bean (vigna radiata L.) serine hydroxymethyltransferase

Abstract

Serine hydroxymethyltransferase, the first enzyme in the pathway for interconversion of C(1) fragments, was purified to homogeneity for the first time from any plant source. The enzyme from 72-h mung bean (Vigna radiata L.) seedlings was isolated using Blue Sepharose CL-6B and folate-AH-Sepharose-4B affinity matrices and had the highest specific activity (1.33 micromoles of HCHO formed per minute per milligram protein) reported hitherto.The enzyme preparation was extremely stable in the presence of folate or l-serine. Pyridoxal 5'-phosphate, ethylenediaminetetraacetate and 2-mercaptoethanol prevented the inactivation of the enzyme during purification. The enzyme functioned optimally at pH 8.5 and had two temperature maxima at 35 and 55 degrees C. The K(m) values for serine were 1.25 and 68 millimolar, corresponding to V(max) values of 1.8 and 5.4 micromoles of HCHO formed per minute per milligram protein, respectively. The K(0.5) value for l-tetrahydrofolate (H(4)folate) was 0.98 millimolar. Glycine, the product of the reaction and d-cycloserine, a structural analog of d-alanine, were linear competitive inhibitors with respect to l-serine with K(i) values of 2.30 and 2.02 millimolar, respectively. Dichloromethotrexate, a substrate analog of H(4)folate was a competitive inhibitor when H(4)folate was the varied substrate. Results presented in this paper suggested that pyridoxal 5'-phosphate may not be essential for catalysis.The sigmoid saturation pattern of H(4)folate (n(H) = 2.0), one of the substrates, the abolition of sigmoidicity by NADH, an allosteric positive effector (n(H) = 1.0) and the increase in sigmoidicity by NAD(+) and adenine nucleotides, negative allosteric effectors (n(H) = 2.4) clearly established that this key enzyme in the folate metabolism was an allosteric protein. Further support for this conclusion were the observations that (a) serine saturation exhibited an intermediary plateau region; (b) partial inhibition by methotrexate, aminopterin, O-phosphoserine, dl-alpha-methylserine and dl-O-methylserine; (c) subunit nature of the enzyme; and (d) decrease in the n(H) value from 2.0 for H(4)folate to 1.5 in presence of l-serine.These results highlight the regulatory nature of mung bean serine hydroxymethyltransferase and its possible involvement in the modulation of the interconversion of folate coenzymes.