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Metabolism and Enzymology

Inhibition of Mung Bean UDP-Glucose: (13)--Glucan Synthase by UDP-Pyridoxal

Evidence for an Active-Site Amino Group

ARCO Plant Cell Research Institute, Dublin, California

UDP-pyridoxal competitively inhibits the Ca²⁺-, cellobiose-activated (13)--glucan synthase activity of unfractionated mung bean (Vigna radiata) membranes, with a K_i of 3.8 ± 0.7 micromolar, when added simultaneously with the substrate UDP-glucose in brief (3 minute) assays. Preincubation of membranes with UDP-pyridoxal and no UDP-glucose, however, causes progressive reduction of the V_max of subsequently assayed enzyme and, after equilibrium is reached, 50% inhibition occurs with 0.84 ± 0.05 micromolar UDP-pyridoxal. This progressive inhibition is reversible provided that the UDP-pyridoxylated membranes are not treated with borohydride, indicating formation of a Schiff's base between the inhibitor and an enzyme amino group. Consistent with this, UDP-pyridoxine is not an inhibitor. The reaction of (13)--glucan synthase with UDP-pyridoxal is stimulated strongly by Ca²⁺ and, less effectively, by cellobiose or sucrose, and the enzyme is protected against UDP-pyridoxal by UDP-glucose or by other competitive inhibitors, implying that modification is occurring at the active site. Pyridoxal phosphate is a less potent and less specific inhibitor. Latent (13)--glucan synthase activity inside membrane vesicles can be unmasked and rendered sensitive to UDP-pyridoxal by the addition of digitonin. Treatment of membrane proteins with UDP-[³H]pyridoxal and borohydride labels a number of polypeptides but labeling of none of these specifically requires Ca²⁺ and sucrose; however, a polypeptide of molecular weight 42,000 is labeled by UDP-[³H]pyridoxal in the presence of Mg²⁺ and copurifies with (13)--glucan synthase activity.

² Current address: Department of Botany, Institute of Life Sciences, The Hebrew University, Jerusalem, Israel.

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