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Articles

Purification and Regulatory Properties of Mung Bean (Vigna Radiata L.) Serine Hydroxymethyltransferase ¹

Department of Biochemistry, Indian Institute of Science, Bangalore 560 012, India

Serine hydroxymethyltransferase, the first enzyme in the pathway for interconversion of C₁ 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°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 (H₄folate) 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 H₄folate was a competitive inhibitor when H₄folate 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 H₄folate (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--methylserine and DL-O-methylserine; (c) subunit nature of the enzyme; and (d) decrease in the n_H value from 2.0 for H₄folate 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.

¹ This investigation was supported from a research grant of the Department of Science and Technology, Government of India, India. D. N. R. is a recipient of the Senior Research Fellowship of the Department of Atomic Energy, Government of India, India.

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