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Butenandt, A. & Karlson, P. (1954) Z. Naturforsch. 9,389-391 Cherbas, L. & Cherbas, P. (1970) Biol. Bull. 138,115-128 Galbraith, M. N. &Horn, D. H. S. (1969) Aust. J. Chem. 22, 1045-1057 Galbraith, M. N., Horn, D. H. S.,Middleton,E. J., Thomson, J. A., Siddall, J. B. & Hafferl, W. (1969~)Chem. Commun. 1134-1135 Galbraith, M. N., Horn, D. H. S., Thomson, J. A., Neufeld, G. J. & Hackney, R. J. (19696) J . Insect Physiol. 15,1225-1233 Gorell, T. A., Gilbert, L. I. &Tash, J. (1972) Insect Biochem.2,94106 Heinrich, G. & Hoffmeister, H. (1970) Z. Naturforsch. 25, 358-361 Hoffman, J. A., Koolman, J., Karlson, P. & Joly, P. (1974) Gen. Comp. Endocrinol.22,90-97 Hoffrneister, H. & Griitzmacher,H. F. (1966) TetrahedronLett. 33,4017-4023 Kaplanis, J. N., Thompson, M. J., Yamarnoto, R. T., Robins, W. E. & Louloudes, S. J. (1966) Steroids 8, 605-623 Karlson, P. &Bode, C. (1969) J. Insect Physiol. 15, 111-118 Karlson, P. & Koolman, J. (1973) Insect Biochem. 3,409-417 Karlson, P., Hoffmeister, K., Hoppe, W. & Huber, R. (1963) Justus Liebigs Ann. Chem. 662,l-20 Karlson, P., Bugany, H., Dopp, H. & Hoyer, G. A. (1972) Hoppe-Seyler’sZ. Physiol. Chem. 353, 1610-1614 King, D. S. (1972) Gen. Comp. Endocrinol. Suppl. 3,221-227 King, D. S. & Siddall, J. B. (1969) Nature (London) 221,955-956 Moriyarna, H., Nakanishi, K., King, D. S., Okauchi, T., Siddall, J. B. & Hafferl, W. (1970) Gen. Comp. Endocrinol. 15,80-87 Ohtaki, T. &Williams, C. M. (1970) Biol. Bull. 138,326-333 Ohtaki, T., Milkman, R. D. &Williams, C. M. (1968) Biol. Bull. 135,322-334 Thompson, M. J., Svoboda,J. A., Kaplanis, J. N. &Robins, W. E. (1972)Proc. Roy. Soc. Ser. B 180; 203-221 Watkinson, I. A. & Clarke, B. S. (1973) Pest Articles & News Summaries 19, 488-506

The Nature of Inhibition and Inactivation of Bovine Liver Glutamate Dehydrogenase by Pyridoxal5’-Phosphate PAUL C. ENGEL and SOO-SE CHEN Department of Biochemistry, University of Shefield, Shefield ,910 2TN, U.K. The well-documented inactivation of bovine liver glutamate dehydrogenase by pyridoxal 5‘-phosphate (Anderson et al., 1967; Piszkiewicz & Smith, 1971; Goldin & Frieden, 1972; Wallis & Holbrook, 1973;Brown et al., 1973) is due to formation of a Schiff base at lysine-126 (Piszkiewicz e t al., 1970). There has been speculation that this residue is essential in binding either substrate or coenzyme, but, if it is essential, its total modification should abolish activity. Goldin & Frieden’s (1972) demonstration that complete inactivation is not achieved even after prolonged incubation with pyridoxal5‘-phosphate called into question the view that lysine-126 is essential. Both Piszkiewicz &Smith (1971) and Brown et al. (1973) attribute the residual activity to unmodified enzyme but fail to account for its persistence. Since the inactivation is reversible by dialysis (Anderson et al., 1967), thereversibility might account for the observed residual activity. Kinetic studies of the inactivation process by Piszkiewicz & Smith (1971) established that a non-covalent glutamate dehydrogenase-pyridoxal 5’-phosphate complex is formed before the Schiff base (see below). These authors omitted the reverse rate constant k-z.

+

G D H PLP

k+i k-1

GDH-PLP Noncova I en t complex

k+i k-2

GDH-PLP Sohiff base

where GDH is glutamate dehydrogenase and PLP is pyridoxal5’-phosphate. We contend that k-2 is not negligible: even if the Schiff base is fully inactive, saturation with pyridoxal 1975

553rd MEETING, LONDON

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k-2 x the initial value. (The nonk+z+k-z covalent complex is assumed to dissociate fully immediately on dilution into an assay mixture.) This explanation of residual activity has now been tested. Glutamate dehydrogenase (0.2mg/ml) was incubated with pyridoxal 5’-phosphate (0.09-7.2m~) in 0.1 M-sodium phosphate, pH7, at 25°C in the dark. Samples (5-10~1) were withdrawn at intervals for fluorimetric assay (Engel & Dalziel, 1969) with 10mMglutamate and ~O,UM-NAD+ at pH7. Activity declined in each case to a minimum within 30-60min, but even with the highest pyridoxal 5’-phosphate concentrations the activity did not fall below 6-7 %of the initialvalue. Pseudo-first-order analysis of the time-courses yielded apparent rate constants which were replotted by the method of Piszkiewicz & Smith (1971) to yield estimates of k+l/k-l (1.59m~)and k+Z(0.59min-’). The reverse rate constant k-z was estimated separately (0.048min-’) from the time-course of reactivation on 100-fold dilution of 90%-inactivated enzyme into buffer. On the basis of these constants a minimum attainable activity of 7.4 %was predicted, very close to the observed values. The final extent of reactivation on dilution was also accurately predicted. Reduction of the Schiff base by NaBH4 renders the inactivation permanent. Our hypothesis suggests that it should be possible to approach complete inactivation of glutamate dehydrogenase by successive cycles of pyridoxal5’-phosphate treatment followed by reduction and dialysis. Treatment in this way with 1.8m~-pyridoxal5‘-phosphate decreased activity by 90% after one cycle and by 99% after two cycles as predicted. (In the absence of pyridoxal5’-phosphate, NaBH4 is without effect.) Absorption measurements at 327nm (Fischer et d.,1963) indicated 1.2mol of modifier incorporated per subunit. It is concluded that complete covalent modification of lysine-126 by pyridoxal 5’phosphate does, after all, totally inactivate glutamate dehydrogenase. Failure to do so in a single treatment merely reflects the equilibrium between the Schiff base and the noncovalent complex. In order to seek the nature of the enzymic malfunction, studies of inhibition and protection were undertaken. Since conversion of the non-covalent glutamate dehydrogenase-pyridoxal5’-phosphate complex into the Schiff base is relatively slow, it is possible to observe steady-state inhibition by pyridoxal 5’-phosphate, due solely to the formation of the non-covalent complex, by adding enzyme to reaction mixtures already containing pyridoxal 5‘-phosphate. The fluorescence of pyridoxal5’-phosphate interfered with fluorimetric assay, and inhibition measurements were therefore made with a Gilford spectrophotometer. Pyridoxal 5‘-phosphate inhibited noncompetitively with respect to 2-oxoglutarate ( ~ O ~ M - N H ~ ~~OPM-NADH) CI, and NADH (50m-NH4CI, 0.5m~-2-oxoglutarate) in 0.1 11M-sodium phosphate, pH 7, at 25°C. This shows that the non-covalent complex is capable of binding both NADH and 2-oxoglutarate. Information about the properties of the covalently modified enzyme were obtained from protection studies. Glutamate dehydrogenase was incubated with 0.6mM-pyridoxal 5’-phosphate in 0.1 M-sodium phosphate, pH7, at 25°C in the presence of 2-oxoglutarate (4, 10, 20,40, 60,8 0 m ~ or ) NADH (0.08,0.10, 0.20, 0.50, 1.00mM). These protecting agents decreased both the rate and final extent of inactivation, but protection by both compounds was partial only, even though saturation by the protecting agents was clearly achieved. Since the inactivation is reversible, it follows that if the covalently modified enzyme is totally unable to bind a compound that can be bound by free enzyme, that compound must be able to protect the enzyme completely against inactivation. The results thus show that binding of NADH and 2-oxoglutarate is hindered, but not prevented, by complete covalent modification at lysine-126. This conflicts with the results of direct binding studies (Brown et al., 1973) which are inherently less sensitive than kinetic methods. Inactivation by pyridoxal 5’-phosphate may reflect: (i) steric or allosteric hindrance of binding or catalysis; (ii) modification of a residue involved in binding; (iii) modification of a catalytic residue. Possibility (ii), as well as (i), remains open since the residual

5‘-phosphate cannot decrease activity below

VOl. 3

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BlOCHEMICAL SOCIETY TRANSACTIONS

binding may be abortive. Nevertheless, the third possibility, namely that lysine126 is directly involved in catalysis rather than binding, merits serious consideration,especially in view of the growing number of other dehydrogenasesshown to be inactivated by pyridoxal 5’-phosphate (Chen & Engel, 1975). The support of the Science Research Council is gratefully acknowledged. Anderson, B. M., Anderson, C. D. & Churchich, J. E. (1967) Biochemistry 5,2893-2900 Brown, A., Culver, J. M. & Fisher, H. F. (1973) Biochemistry 12,4367-4373 Chen, S.-S. & Engel, P. C. (1975) Biochem. Soc. Trans.3,80432 Engel, P. C. & Dalziel, K. (1969) Biochem.J. 115, 621-631 Fischer, E. H., Forrey, A. W., Hedrick, J. L., Hughes, R. C., Kent, A. B. & Krebs, E. G. (1963) in ChemicalandBiological Aspects OfPyridoxal Catalysis (Snell, E. E., Fasella, P. M., Braunstein, A. & Rossi-Fanelli, A., eds.), p. 543, Pergamon Press, Oxford Goldin, B. R. & Frieden, C. (1972) J. Biol. Chem. 247,2139-2144 Piszkiewicz, D. & Smith, E. L. (1971) Biochemistry 10,4544-4552 Piszkiewicz, D., Landon, M. & Smith, E. L. (1970) J. Biol. Chem. 245,2622-2626 Wallis, R. B. & Holbrook, J. J. (1973) Biochem.J. 133,173-182

Inactivation of Nicotinamide-Adenine Dinucleotide-Link6dDehydrogenases by Pyridoxal 5’-Phosphate SOO-SE CHEN and PAUL C. ENGEL Department of Biochemistry, University of Shefield, Shefield S10 2TN, U.K. Pyridoxal5’-phosphate has been used as a specific reagent for modifying essential lysine residues in various enzymes including glyceraldehyde 3-phosphate dehydrogenase (Ronchi et al., 1969) horse liver alcohol dehydrogenase (McKinley-McKee & Morris, 1972) and various glycolyticenzymes [referencesin Colombo & Marcus (1974)l. A single treatment never totally abolishes activity, however. This could mean that the lysine residues are not, after all, strictly essential. Alternatively, however, as shown for glutamate dehydrogenase(Engel & Chen, 1975), the Schiff base may be in equilibrium with a non-covalent enzymemodifier complex which rapidly dissociates on dilution. Mechanistic and structural features in common may be expected among NAD(P)+linked dehydrogenases (references in Everse et al., 1971) and similarities in threedimensional structure have indeed been found (Buehner et al., 1973). Homology has been noted between the sequences around lysine-212 in glyceraldehyde 3-phosphate dehydrogenase and lysine-126 in glutamate dehydrogenase,both residues that are specifically modified by pyridoxal 5’-phosphate (Smith et al., 1970; Forcina et al., 1971). It seemed possible that an essential lysine might be present in other NAD(P)+-linked dehydrogenases. Accordingly, the effects of pyridoxal5‘-phosphate on horse liver alcohol dehydrogenase and glyceraldehyde 3-phosphate dehydrogenase were re-examined and effects on pig heart mitochondria1 malate dehydrogenase, hog muscle M4 lactate dehydrogenase and yeast alcohol dehydrogenase were also studied. The main results are summarized in Table 1. At 10°C (0.1M-potassium phosphate, pH8), the maximum inactivation of horse liver alcohol dehydrogenase attainable in one cycle of pyridoxal5’-phosphate treatment was 7 4 7 5 %, and 2-3 mol of pyridoxal 5’-phosphatewasincorporated per mol of enzyme subunit. Reduction by NaBHc rendered the inactivation irreversible.After dialysis, a second pyridoxal5’-phosphate treatment decreased the residual activity by 75 %, and after three cycles only 1-2% of the original activity remained. A detailed kinetic and equilibrium analysis of single-cycle inactivation and reactivation on dilution also produced results consistent with an equilibrium between inactive Schiff base and non-covalent complex (cf. McKinley-McKee & Morris, 1972). As an instantaneous steady-state inhibitor (Engel & Chen, 1975) pyridoxal5‘-phosphate was non-competitive with respect both to NADH and acetaldehyde.In protection experiments,however, NAD+ and NADH both protected completely against pyridoxal 5’-phosphate inactivation, whereas ethanol and acetaldehyde gave no protection. 1975