612
BIOCHEMICAL SOCIETY TRANSACTIONS
I thank Mrs. Christine Eggleton for her excellent technical assistance.
Gibson, K. D., Neuberger, A. & Tait, G. H. (1962) Biochem. J. 84,483-490 Greenberg, D. M. (1969) Metab. Pathways, 3rd Ed. 3,237-315 Herbst, E. J., Keister, D. L. &Weaver, R. H. (1958) Arch. Biochem. Biophys. 75, 178-185 Neuberger, A. & Tait, G. H. (1962)J. Chem. SOC.3963-3968 Neuberger, A., Sandy, J. D. & Tait, G. H. (1973) Biochem. J. 136,477-490 Poso, H., Hannonen, P., Hirnberg, J.-J. & Janne, J. (1976) Biochem. Biophys. Res. Commun. 68, 227-234 Russell, D. H., Medina, V. J. & Snyder, S. H. (1970)J. Biol. Chem. 245,6732-6738 Tabor, H. & Tabor, C. W. (1972) Adv. Enzymol. Relat. Areas Mol. Biol. 36,203-268 Tait, G . H. (1975) Biochem. J. 146,191-204
Biphasic Interactions between a Neurospora crassa Glutamate Dehydrogenase and Reduced NicotinamideAdenine Dinucleotide Phosphate M. G. GORE* and M. IWATSUBOt D,partment of Physiology and Biochemistry, University of Southampton, Southampton SO9 3TU, U.K.,and f Centre de Genetique Moleculaire, C.N.R.S.,Gij-sur- Yvette, France
The NADP+-specific glutamate dehydrogenase (EC 1.4.1.4) from wild-type Neurosporu crassa has been shown to exist in a predominantly active conformation in solutions above pH 7.1 and in an inactive conformation at more acid pH values. The presence of NADPH shifts the equilibrium towards the inactive conformation (West et al., 1967). This enzyme has been shown by fluorimetric studies (at equilibrium) to form a stable binary complex with NADPH (Gore et al., 1972). Further studies (Gore & Iwatsubo, 1975) have suggested that the system is unstable and that the original binary complex formed, for example, at pH 7.5 undergoes a structural transition before attaining an equilibrium state. The studies by Gore & Iwatsubo (1975) have shown that a structural transition of reasonable magnitude(80kJ/mol) will take place after the formation of an initial binary complex when a solution of enzyme in the active conformation is rapidly mixed with NADPH at pH values between 6.5 and 8.5. The signal change from this presumed conformational change had a maximum amplitude when enzyme at pH 8.0 or above was mixed 1 :6 (mol/mol) withasolution ofNADPH at pH6.5. Since theseare theconditions that would cause a maximum loss in catalytic activity of the enzyme, it is suggested either that as the enzyme assumed the inactive conformation it obtained a greater affinity for NADPH or that a change in the configuration of the enzyme-bound NADPH took place altering its spectral properties. The cause of this signal change has been investigated to try and identify thenature of the control mechanismexerted by NADPH. By use of a solution of enzyme preincubated in IOm-Tris/HCl buffer, pH8.1, and mixing this with a sixfold excess of NADPH in 50mM-pOta~iUmphosphate buffer, pH6.5, it was possible to observe the binding of NADPH to the active conformation at pH 6.5 where the inactive conformation normally prevails. Alternatively, it was possible to treat the inactive conformation (enzyme preincubated at pH6.5) with an excess of NADPH in Tris/HCI buffer, pH8.5 (final pH8.1), thus momentarily trapping the inactive conformation in an environment normally associated with the catalytically active species of the enzyme. A rapid formation of a binary complex took place in both situations, followed by a slower relaxation of enzyme conformation to that which would normally predominate in the new environment. A typical situation is demonstrated in ~ of active enzyme in IOmM-Tris/HCl buffer, pH8.1, was Fig. 1. Here a 1 4 p solution mixed with a solution of NADPH ( 3 0 ~ to ~ give ) a final pH of 6.5. The rapid initial binding was analysed as a first-order process k,,,. (s-’) when the final NADPH concentration was at least 10 times that of the enzyme. The rate, k,,,. (s-l), was then plotted against the final concentration of NADPH used, and the slope of this graph described the ‘on-rate’ of NADPH to the binary complex. Values for k-, i.e. the ‘off-rate’ of 1976
613
563rd MEETING, LONDON
I20
I
I
+
.
Time fms) . - I
I
I
I =I
i
I I
1 - 1I
I
I
I
II ---I I
I
-
I
1000 mr
t
I
Fig. 1. Change influorescence at 350nm when Neurospora crassa glutamate dehydrogenase solution ( 1 4 ~ in ~ ) 10mM-Tris/HCl bufer, pH8.1, was mixed 1:6 (mollmol) wirh NADPH ( 3 0 , ~ in ~ )50mM-potassium phosphate buffer, pH6.5 The final pH was 6.5 at 10°C. The inset is a logarithmic plot of 100 minus the percentage change in signal (S) in trace A at various times (ms). Trace A was obtained by using a calibration of 20ms per horizontal division, and trace B was obtained by using identical conditions except that the time-scale used was 1000ms/horizontal division. The arrow indicates the origin of both traces. Trace C was the signal at the end of the reaction. NADPH from the binary complex in various conditions, were obtained by diluting a preformed binary complex by a tenfold excess of various buffers. The subsequent decay of the binary complex could be monitored by changes in nucleotide fluorescence and the value k- (s-') calculated. Since k J k + equals the dissociation constant (&) for the equilibrium between the NADPH and the binary complex, it was possible to estimate the relative interactions of various reactants. From the values of Kd calculated from values of k- (s-') and k+ (M-'-s-') it became clear that inactive enzyme has a higher affinity for NADPH than does active enzyme at pH6.5. At this pH all of the enzyme present would normally be inactive. However, at pH8.1 there is little difference between the affinities of inactive enzyme for NADPH or active enzyme for NADPH, and both equilibria are governed by much larger dissociation constants. This is in good agreement with results obtained by spectrofluorimetry (Gore et al., 1972). Thus it seemed probable that the relatively slow fluorescencechange already described (Gore &Iwatsubo, 1975)is due to a secondary phase of NADPH binding to the enzyme, which obtains a higher affinity for NADPH as it assumes the inactive conformation. It is also probable that this secondary binding process is taking place between NADPH and enzyme already partially saturated by the reduced coenzyme. The rate-limiting step is therefore k+l in the equilibrium: kti
E,*NADPH
1 . E,*NADPH k-1
Vol. 4
614
BIOCHEMICAL SOCIETY TRANSACTIONS
The rate constant for this reaction (trace B ) is 20.7min-L at 26°C and is approximately 100 times faster than the reaction rates controlling the equilibrium between E, and El (Ashby et al., 1974). The rate of reaction described by k+l in the equilibrium above was not affected by changes in NADPH concentration or small changes in ionic strength. Binding studies at equilibrium have suggested that there is no strong co-operativity between NADPH-binding sites at, e.g., pH7.5 (Gore et al., 1972). Ashby, B., Wootton, J. C. & Fincharn, J. R. S. (1974) Biochem. J . 143,317-329 Gore, M. G. & Iwatsubo, M. (1975) Biochem. J . 147, 181-184 Gore, M. G., Greenwood, C. & Holbrook, J. J. (1972) Biochem. J . 1 2 7 , 3 0 ~ - 3 1 ~ West, D. J., Tuveson, R. W., Barratt, R. W. & Fincharn, J. R. S. (1967) J. Biol. Chem. 242, 21 34-2142
Mandelate Dehydrogenase with Altered Stereospecificity in Mutant Strains of Acinetobacter calcoaceticus N.C.I.B. 8250 C. A. FEWSON, JEAN D. BEGGS,* ELLEN SEENAN and E. F. AHLQUISTt Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Several micro-organisms can use mandelate as the sole source of carbon and energy for growth; some can use both isomers, others only one (Hegeman et al., 1970). Acinetobactev calcoaceticus N.C.I.B. 8250 has an inducible stereospecific L(+)mandelate dehydrogenase and grows on L(+)-mandelate, but not on D(-)-mandelate (Kennedy & Fewson, 1968a,b). We have now isolated mutant strains able to grow on both isomers or on D(-)-mandelate but not on L(+)-mandelate. Mutant strains able to use both isomers were selected on the basis of ability to grow on D(-)-mandelate. Some of the mutants arose spontaneously, and others appeared after treatment with N-methyl-N’-nitro-N-nitrosoguanidine.Much of our work has been done with strain 41, which appears to be typical of most of the mutant strains isolated; they are all identical with strain N.C.I.B. 8250, except for the additional ability to use D(-)-mandelate as source of carbon and energy for growth. Strains able to use D(-)-mandelate, but not L(+)-mandelate, were constructed by transformation, by methods based on those described by Juni (1972) and Sawula & Crawford (1972). DNA was isolated from strain 41 and mixed with strain N F 1408 [which lacks L(+)-mandelate dehydrogenase; Livingstone & Fewson, 19721on agar plates containing D(-)-mandelate and a trace of phenylglyoxylate to serve as inducer. Most of the transformants produced (e.g. strain 41 Z) could grow on both isomers of mandelate, but a few (e.g. strain D40G) could grow on only D(-)-mandelate and had no L(+)-mandelate dehydrogenase activity. It appears that strain 41 can utilize mandelate by means of a new dehydrogenase specific for D(-)-mandelate. There was no evidence for mandelate racemization in experiments using methods that could detect the interconversion of the two isomers in Pseudomonas putida N.C.I.B. 9494. Construction of strain D40G rules out the involvement of L(+)-mandelate dehydrogenase in the oxidation of D(-)-mandelate. Extracts of strains 41 and D40G decoloured 2,6-dichlorophenol-indophenoland other dyes in the presence of D(-)-mandelate. Phenylglyoxylate formation in extracts oxidizing D(-)mandelate was confirmed by formation of the 2,4-dinitrophenylhydrazone. D(-)-Mandelate dehydrogenase activity was induced by phenylglyoxylate in strains 41, D40G and 412. Maximum D(-)-mandelate dehydrogenase activity was never more than 20 %of the maximum L(+)-mandelate dehydrogenase activity observed in the parent strain N.C.I.B. 8250 or in strain 41. This is probably a reflexion of the activity in uiuo * Present address: Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, U.K.
t Present address: Biological Laboratories, University of Kent, Canterbury CT2 7NJ, U.K. 1976
BIOCHEMICAL SOCIETY TRANSACTIONS
I thank Mrs. Christine Eggleton for her excellent technical assistance.
Gibson, K. D., Neuberger, A. & Tait, G. H. (1962) Biochem. J. 84,483-490 Greenberg, D. M. (1969) Metab. Pathways, 3rd Ed. 3,237-315 Herbst, E. J., Keister, D. L. &Weaver, R. H. (1958) Arch. Biochem. Biophys. 75, 178-185 Neuberger, A. & Tait, G. H. (1962)J. Chem. SOC.3963-3968 Neuberger, A., Sandy, J. D. & Tait, G. H. (1973) Biochem. J. 136,477-490 Poso, H., Hannonen, P., Hirnberg, J.-J. & Janne, J. (1976) Biochem. Biophys. Res. Commun. 68, 227-234 Russell, D. H., Medina, V. J. & Snyder, S. H. (1970)J. Biol. Chem. 245,6732-6738 Tabor, H. & Tabor, C. W. (1972) Adv. Enzymol. Relat. Areas Mol. Biol. 36,203-268 Tait, G . H. (1975) Biochem. J. 146,191-204
Biphasic Interactions between a Neurospora crassa Glutamate Dehydrogenase and Reduced NicotinamideAdenine Dinucleotide Phosphate M. G. GORE* and M. IWATSUBOt D,partment of Physiology and Biochemistry, University of Southampton, Southampton SO9 3TU, U.K.,and f Centre de Genetique Moleculaire, C.N.R.S.,Gij-sur- Yvette, France
The NADP+-specific glutamate dehydrogenase (EC 1.4.1.4) from wild-type Neurosporu crassa has been shown to exist in a predominantly active conformation in solutions above pH 7.1 and in an inactive conformation at more acid pH values. The presence of NADPH shifts the equilibrium towards the inactive conformation (West et al., 1967). This enzyme has been shown by fluorimetric studies (at equilibrium) to form a stable binary complex with NADPH (Gore et al., 1972). Further studies (Gore & Iwatsubo, 1975) have suggested that the system is unstable and that the original binary complex formed, for example, at pH 7.5 undergoes a structural transition before attaining an equilibrium state. The studies by Gore & Iwatsubo (1975) have shown that a structural transition of reasonable magnitude(80kJ/mol) will take place after the formation of an initial binary complex when a solution of enzyme in the active conformation is rapidly mixed with NADPH at pH values between 6.5 and 8.5. The signal change from this presumed conformational change had a maximum amplitude when enzyme at pH 8.0 or above was mixed 1 :6 (mol/mol) withasolution ofNADPH at pH6.5. Since theseare theconditions that would cause a maximum loss in catalytic activity of the enzyme, it is suggested either that as the enzyme assumed the inactive conformation it obtained a greater affinity for NADPH or that a change in the configuration of the enzyme-bound NADPH took place altering its spectral properties. The cause of this signal change has been investigated to try and identify thenature of the control mechanismexerted by NADPH. By use of a solution of enzyme preincubated in IOm-Tris/HCl buffer, pH8.1, and mixing this with a sixfold excess of NADPH in 50mM-pOta~iUmphosphate buffer, pH6.5, it was possible to observe the binding of NADPH to the active conformation at pH 6.5 where the inactive conformation normally prevails. Alternatively, it was possible to treat the inactive conformation (enzyme preincubated at pH6.5) with an excess of NADPH in Tris/HCI buffer, pH8.5 (final pH8.1), thus momentarily trapping the inactive conformation in an environment normally associated with the catalytically active species of the enzyme. A rapid formation of a binary complex took place in both situations, followed by a slower relaxation of enzyme conformation to that which would normally predominate in the new environment. A typical situation is demonstrated in ~ of active enzyme in IOmM-Tris/HCl buffer, pH8.1, was Fig. 1. Here a 1 4 p solution mixed with a solution of NADPH ( 3 0 ~ to ~ give ) a final pH of 6.5. The rapid initial binding was analysed as a first-order process k,,,. (s-’) when the final NADPH concentration was at least 10 times that of the enzyme. The rate, k,,,. (s-l), was then plotted against the final concentration of NADPH used, and the slope of this graph described the ‘on-rate’ of NADPH to the binary complex. Values for k-, i.e. the ‘off-rate’ of 1976
613
563rd MEETING, LONDON
I20
I
I
+
.
Time fms) . - I
I
I
I =I
i
I I
1 - 1I
I
I
I
II ---I I
I
-
I
1000 mr
t
I
Fig. 1. Change influorescence at 350nm when Neurospora crassa glutamate dehydrogenase solution ( 1 4 ~ in ~ ) 10mM-Tris/HCl bufer, pH8.1, was mixed 1:6 (mollmol) wirh NADPH ( 3 0 , ~ in ~ )50mM-potassium phosphate buffer, pH6.5 The final pH was 6.5 at 10°C. The inset is a logarithmic plot of 100 minus the percentage change in signal (S) in trace A at various times (ms). Trace A was obtained by using a calibration of 20ms per horizontal division, and trace B was obtained by using identical conditions except that the time-scale used was 1000ms/horizontal division. The arrow indicates the origin of both traces. Trace C was the signal at the end of the reaction. NADPH from the binary complex in various conditions, were obtained by diluting a preformed binary complex by a tenfold excess of various buffers. The subsequent decay of the binary complex could be monitored by changes in nucleotide fluorescence and the value k- (s-') calculated. Since k J k + equals the dissociation constant (&) for the equilibrium between the NADPH and the binary complex, it was possible to estimate the relative interactions of various reactants. From the values of Kd calculated from values of k- (s-') and k+ (M-'-s-') it became clear that inactive enzyme has a higher affinity for NADPH than does active enzyme at pH6.5. At this pH all of the enzyme present would normally be inactive. However, at pH8.1 there is little difference between the affinities of inactive enzyme for NADPH or active enzyme for NADPH, and both equilibria are governed by much larger dissociation constants. This is in good agreement with results obtained by spectrofluorimetry (Gore et al., 1972). Thus it seemed probable that the relatively slow fluorescencechange already described (Gore &Iwatsubo, 1975)is due to a secondary phase of NADPH binding to the enzyme, which obtains a higher affinity for NADPH as it assumes the inactive conformation. It is also probable that this secondary binding process is taking place between NADPH and enzyme already partially saturated by the reduced coenzyme. The rate-limiting step is therefore k+l in the equilibrium: kti
E,*NADPH
1 . E,*NADPH k-1
Vol. 4
614
BIOCHEMICAL SOCIETY TRANSACTIONS
The rate constant for this reaction (trace B ) is 20.7min-L at 26°C and is approximately 100 times faster than the reaction rates controlling the equilibrium between E, and El (Ashby et al., 1974). The rate of reaction described by k+l in the equilibrium above was not affected by changes in NADPH concentration or small changes in ionic strength. Binding studies at equilibrium have suggested that there is no strong co-operativity between NADPH-binding sites at, e.g., pH7.5 (Gore et al., 1972). Ashby, B., Wootton, J. C. & Fincharn, J. R. S. (1974) Biochem. J . 143,317-329 Gore, M. G. & Iwatsubo, M. (1975) Biochem. J . 147, 181-184 Gore, M. G., Greenwood, C. & Holbrook, J. J. (1972) Biochem. J . 1 2 7 , 3 0 ~ - 3 1 ~ West, D. J., Tuveson, R. W., Barratt, R. W. & Fincharn, J. R. S. (1967) J. Biol. Chem. 242, 21 34-2142
Mandelate Dehydrogenase with Altered Stereospecificity in Mutant Strains of Acinetobacter calcoaceticus N.C.I.B. 8250 C. A. FEWSON, JEAN D. BEGGS,* ELLEN SEENAN and E. F. AHLQUISTt Department of Biochemistry, University of Glasgow, Glasgow G12 8QQ, Scotland, U.K.
Several micro-organisms can use mandelate as the sole source of carbon and energy for growth; some can use both isomers, others only one (Hegeman et al., 1970). Acinetobactev calcoaceticus N.C.I.B. 8250 has an inducible stereospecific L(+)mandelate dehydrogenase and grows on L(+)-mandelate, but not on D(-)-mandelate (Kennedy & Fewson, 1968a,b). We have now isolated mutant strains able to grow on both isomers or on D(-)-mandelate but not on L(+)-mandelate. Mutant strains able to use both isomers were selected on the basis of ability to grow on D(-)-mandelate. Some of the mutants arose spontaneously, and others appeared after treatment with N-methyl-N’-nitro-N-nitrosoguanidine.Much of our work has been done with strain 41, which appears to be typical of most of the mutant strains isolated; they are all identical with strain N.C.I.B. 8250, except for the additional ability to use D(-)-mandelate as source of carbon and energy for growth. Strains able to use D(-)-mandelate, but not L(+)-mandelate, were constructed by transformation, by methods based on those described by Juni (1972) and Sawula & Crawford (1972). DNA was isolated from strain 41 and mixed with strain N F 1408 [which lacks L(+)-mandelate dehydrogenase; Livingstone & Fewson, 19721on agar plates containing D(-)-mandelate and a trace of phenylglyoxylate to serve as inducer. Most of the transformants produced (e.g. strain 41 Z) could grow on both isomers of mandelate, but a few (e.g. strain D40G) could grow on only D(-)-mandelate and had no L(+)-mandelate dehydrogenase activity. It appears that strain 41 can utilize mandelate by means of a new dehydrogenase specific for D(-)-mandelate. There was no evidence for mandelate racemization in experiments using methods that could detect the interconversion of the two isomers in Pseudomonas putida N.C.I.B. 9494. Construction of strain D40G rules out the involvement of L(+)-mandelate dehydrogenase in the oxidation of D(-)-mandelate. Extracts of strains 41 and D40G decoloured 2,6-dichlorophenol-indophenoland other dyes in the presence of D(-)-mandelate. Phenylglyoxylate formation in extracts oxidizing D(-)mandelate was confirmed by formation of the 2,4-dinitrophenylhydrazone. D(-)-Mandelate dehydrogenase activity was induced by phenylglyoxylate in strains 41, D40G and 412. Maximum D(-)-mandelate dehydrogenase activity was never more than 20 %of the maximum L(+)-mandelate dehydrogenase activity observed in the parent strain N.C.I.B. 8250 or in strain 41. This is probably a reflexion of the activity in uiuo * Present address: Department of Molecular Biology, University of Edinburgh, Edinburgh EH9 3JR, Scotland, U.K.
t Present address: Biological Laboratories, University of Kent, Canterbury CT2 7NJ, U.K. 1976
