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1278 BIOCHEMICAL SOCIETY TRANSACTIONS Studies on the Reactivity towards Pyridoxall 5’-Phosphate of a Neurospora Glutamate Dehydrogenase MICHAEL G. GORE, JOHN C. WOOTTON and JOHN R. S. FINCHAM Department of Genetics, University of Leeds, Leeds LS2 9JT, U.K. Some fragments of the amino acid sequence of the NADP-dependent glutamate dehydrogenase of Neurospora crussa show significant homology with parts of the sequence of the bovine enzyme (Wootton et al., 1973). These include a 30-residue fragment that aligns with residues 105-134 of the bovine enzyme sequence described by Moon et al. (1972), including a lysine residue corresponding to lysine-126, which in the bovine enzyme is highly reactive towards pyridoxal 5‘-phosphate (Piszkiewicz et al., 1970). This homology prompted us to study the reactivity of the Neurospora enzyme towards pyridoxal 5’-phosphate, Two modes of binding of pyridoxal 5’-phosphate to the enzyme can be detected at pH7.6 in phosphate buffer. The addition of enzyme to a solution of pyridoxal 5‘-phosphate causes a time-dependent change in the absorption spectrum of the pyridoxal 5‘-phosphate. There is a rapid decreasein absorbanceat approx. 375nm, probably due to non-specificbinding that decreases the amount of hydrated and free aldehyde in solution (Avramovic-Zikic& Madsen, 1972), and an increase in absorbance at approx. 425nm, probably due to the formation of a Schiff base. The addition of NaBH4 to the mixture results in the formation of a reduced Schiff base, which is stable to dialysis and has an absorbance peak at approx. 325nm. The formation of this stable complex is not necessarily at the same rate as the change in the absorption spectrum of the pyridoxal 5‘-phosphate. It is probable that a number of low-affinitysites are rapidly occupied, causing the decreasein absorbance at 375nm, and that Schiff-base formation takes place at a lower rate. The stoicheiometry of Schiff-base formation is consistent with one highly reactive site and one or more other sites able to react although at much lower rates under the conditions used (100m-potassium phosphate buffer, pH7.6, at 21°C; a 60-fold excess of pyridoxal 5‘-phosphate was added to a solution of 20p~-enzymemonomer, calculated on a subunit molecular weight of 50000). The initial rapid Schiff-base formation occurs at the same rate as a decrease in the enzyme activity in both directions. Under the above conditions the inactivation process almost ceases after about 5min, at which time the enzyme activity is diminished to about 40 % of the original and approx. 1mol of pyridoxal5’-phosphate is bound/mol of subunit. When the pyridoxal5’-phosphate concentration is not limiting (200-fold molar excess over enzyme subunits) this inactivation process follows first-order kinetics (rate constant 0.12min-’). This is the same rate as a conformation change, which can be followed by a change in protein fluorescence, from an active to an inactive state occurring when the enzyme alone is transferred from pH8.0 to 7.0 (first-order rate constant of the fluorescencechangeand theconcomitant inactivationprocess is 0.13min-’ at 21°C; B. Ashby, unpublished work). It is possible therefore that the group highly reactive towards pyridoxal5‘-phosphate is inaccessible in the active conformation of the enzyme, and that the reaction observed is with an inactive conformation of the enzyme and the rate of the reaction is limited by the slow conformation change from the acfiveto the inactive state. Avramovic-Zikic, 0. & Madsen, N. B. (1972) J. Biol. Chem. 247, 6999-7004 Moon, K., Piszkiewicz, D. &Smith,E. L. (1972) Proc. Nut. Acad. Sci. U.S.69,1380-1384 Piszkiewicz, D., Landon, M. & Smith, E. L. (1970) J. Biol. Chem. 245,2622-2626 Wootton, J. C., Chambers, G. K., Taylor, J. C. & Fincham, J. R. S. (1973) Nature (London) New Biol. 241,4243 1973