A Kinetic Study of Glucose-6-phosphate Dehydrogenase

A Kinetic Study of Glucose-6-phosphate Dehydrogenase (Received for publication, September 10, 1975) MOHAMMED. KANJ, MYRON L. TOEWS, AND W. ROBERT CARPER* From the Department of Chemistry, Wichita State University, Wichita, Kansas 67208 The steady state kinetics of pig liver glucose-6-phosphate dehydrogenase is consistent with an ordered, sequential mechanism in which NADP is bound first and NADPH released last. K,, is 9.0 PM, K, is 4.8 PM, and K, is 36 pm. Glucosamine 6-phosphate, a substrate analogue and competitive inhibitor, is used to help rule out a possible random mechanism. ADP is seen to form a complex with the free form of the enzyme whereas ATP forms a complex with both the free and E.NADP forms of the enzyme. The K, for the E.ADP complex is 1.9 mm, while the K, values for the E ATP and E NADP ATP complexes are 7.2 and 4.5 mm, respectively. The kinetic mechanism of glucose-6-phosphate dehydrogenase has been studied from several sources including Candida utilis (l), human erythrocyte (Z), Leuconosto mesenteroides (3), human blood platelets (4), and rat liver (5). All of the above yield results which are consistent with an ordered, sequential mechanism in which NADP+ is bound first and NADPH released last. Furthermore, glucose-6-phosphate dehydrogenase from human erythrocytes and C. utilis give sigmoid kinetics when NADP+ concentration is varied. Studies on the steady state mechanism of this enzyme from all sources are necessarily limited by the inability to characterize the reverse reaction due to the marked instability of gluconolactone 6-phosphate. n this work, the glucose-6-phosphate dehydrogenase used for kinetics was purified as described in the preceding paper (5). n this study, initial velocity patterns are used to rule out a ping-pong mechanism, while product inhibition studies and the use of a competitive inhibitor rule out a rapid equilibrium random mechanism with dead-end complexes. The use of a competitive inhibitor further established the order of substrate addition with NADP+ and glucose 6-phosphate being the first and second substrates, respectively. ATP and ADP inhibition studies have been done on glucose- B-phosphate dehydrogenase from human blood platelets (4). However, the author does not state which enzyme form ADP combines with while he indicates that ATP combines with both the E and E.NADP+ forms of the enzyme. n this work, inhibition studies using ADP show that it competes with NADP+ for the free enzyme, whereas ATP combines with both the free and E.NADP+ forms of the enzyme. MATERALS AND METHODS All chemicals used were purchased as described under Materials and Methods in the preceding paper (5). n addition, the disodium salts of glucosamine 6.phosphate and ATP were purchased from Sigma *To whom all communications regarding this paper should be addressed. Chemical Co. The monosodium salt of ADP was purchased from Calbiochem. The reaction was followed by observing the increase in optical density at 340 nm with the production of NADPH. RESULTS Data Analysis-n the notation of Cleland (6) the Ordered Bi Bi mechanism can be depicted as follows: (A) (El P (Q) + + i E (11 (LA) EAB) (EQ (EPQ) where E, A, B, P, Q represent enzyme, NADP+, glucose 6-phosphate, 6-phosphogluconolactone, and NADPH, respectively. Data from the initial velocity studies were fitted to Equation 2: where V, is the maximum velocity, K, and K, represent the Michaelis constants for NADP+ and glucose 6-phosphate, respectively, and K,, is the E.NADP+ complex dissociation constant. For product inhibition studies, Equation 3 was used: VAB Y = L% * KaB + \A + AB * Q(Kla\ + KaBl/K 19 (3) where K,,, K,,, K,, A, B, and V, are as defined above, Q is the NADPH concentration, and K,, is the E.NADPH dissociation constant. For dead-end inhibition studies, the basic equation used vvas: = L,*B,[K,aKb(l t W) + KaB(l *,K) + KbA(l *,K l + AB] (41 Where Z is the inhibitor concentration, and K, and K, are inhibition constants. f the inhibitor combines with the free form of the enzyme, as with ADP, the K, term appears. Alternately, if the inhibitor combines with the EA (E.NADP+) form of the enzyme, the K, term will arise. Finally, if both 2258

effects occur, Equation 4 will describe the total process as in the case of ATP. Equation 4 is readily derived from King- Altman diagrams (7) and the papers of Cleland (6,8). nitial Velocity Studies-For initial velocity studies Equation 2 was used in the form of: Glucose-6-P Dehydrogenase Kinetics The Lineweaver-Burk (9) plot of reciprocal velocity uersus reciprocal concentration of glucose 6-phosphate at various fixed concentrations of NADP+ gives a family of lines which intersect at a common point as shown in Fig. 1. This rules out any mechanism which requires the dissociation of one product before the addition of the second substrate and is thus a sequential mechanism. The data were analyzed using a modified version of Cleland s program (10) in which all points were given equal weight (11). The Michaelis constants are 4.8 (&0.5) FM for NADP and 36 (~3) FM for glucose B-phosphate. The dissociation constant for NADP+, K,,, is 9.0 (10.8) PM, and V, is 0.0059 gm/min. These results may be compared with the results reported for the human platelet enzyme (4), where K,, is 6 pm, K, is 12 pm, and K, is 6 FM. Product nhibition Studies-n 1963, Cleland (8) clearly pointed out how product inhibition patterns are capable of demonstrating the order of addition of substrates in a Sequential Bi Bi mechanism. n this study, when NADP was varied and NADPH was the inhibitor, Equation 3 was used in the following form: When glucose 6-phosphate was varied and NADPH was the inhibitor, Equation 3 was used in the form of: Fig. 2 shows the result of fitting Equation 6 to data collected in an experiment in which the concentrations of both NADP+ and NADPH were varied. The inhibition pattern is competitive and similar to that observed by others (2-4). The parameters, K,, K,,, and K, were used to calculate a K,, value of 11 pm from the replot of the slope in Fig. 3. Further evidence for a sequential mechanism was also observed when saturated levels of glucose 6-phosphate, and varied levels of NADP and NADPH also exhibited competitive inhibition. Fig. 4 shows the result of fitting Equation 7 to data in an experiment where glucose 6-phosphate was the variable substrate at several NADPH concentrations. A noncompetitive product inhibition pattern was observed and the dissociation constant, K,,, was calculated to be 7 PM from a replot of the slopes from Fig. 4 in Fig. 5. From these results we will temporarily conclude that K,, is 9 (12) FM. n addition to this experiment, we also obtained additional evidence for the ordered addition of substrates when no inhibition was observed The abbreviations and symbols used are those recommended by UPAC-UB (12). 2Figs. 3, 5, 7, 13, and 14 are presented in miniprint format immediately following this paper. For the convenience of those who prefer to obtain them in the form of full size photocopies, these same data are available as JBC Document No. 75M-1241. Orders should specify the title, authors, and reference to this paper and the JBC Document Number, and the number of copies desired. Orders should be addressed to The Journal of Biological Chemistry, 9650 Rockville Pike, Bethesda, Md. 20014, and must be accompanied by a remittance to the order of the Journal in the amount of $1.00. (6) FG. 1. nitial velocity pattern with glucose 6-phosphate (Glc-6-P) as the varied substrate at 30. Each sample contained 4 x 10m3 units of enzyme (0.0081 FM), 50 mm NaOH/glycine buffer, ph 8.5, and 1 mm M&l,. -0.O 0.1 FG. 2. NADPH inhibition plot at 30. Glucose 6-phosphate is 130 ELM, NADP+ varied from 10 to 50 PM, and NADPH varied from 12.5 to 50 /M. E, = 0.0081 /AM.,,M 0.O.01.03 /Glc-6-P, FG. 4. NADPH inhibition plot at 30 with glucose 6.phosphate (Glc-6-P) as the variable substrate. NADP+ concentration is 25 PM, glucose 6-phosphate varied from 30 to 130 KM, and NADPH varied from 20 to 100 @M. E, = 0.0081 PM. at saturation levels of NADP+ and with varied levels of glucose 6-phosphate and NADPH. n summary, the inhibition patterns are consistent with an Ordered Bi Bi mechanism where the coenzyme adds first and is released last from the enzyme. However, these inhibition patterns do not rule out a Theorell-Chance and rapid equilibrium random mechanism with dead-end complexes such as E.NADP+.6-phosphogluconolactone. Product inhibition studies using 6-phospho-&gluconolactone are impractical due to the instability of the compound in aqueous solution (13). Dead End nhibition Studies-n 1964, Fromm (14) showed how competitive inhibitors might be used to distinguish between ordered and random mechanisms for two substrate enzymes. A second advantage of using compounds which compete with a substrate for the same enzymic site in ordered,m

2260 Glucose-6-P Dehydrogenase Kinetics systems is that the order of substrate addition can be determined. Glucosamme 6-phosphate has been shown to be a competitive inhibitor with respect to glucose 6-phosphate in rat liver glucose-6-phosphate dehydrogenase reaction (15). The same effect was observed with pig liver glucose-6-phosphate dehydrogenase and thus glucosamine 6-phosphate was used as a substrate analogue for glucose 6-phosphate in an attempt to form a dead-end complex in the following manner: E.NADP+ + glucosamine B-phosphate z E.NADP+.glucosamine 6.phosphate (8) For the above studies Equation 4 was used in the following ways. When NADP+ was the variable substrate and glucosamine 6-phosphate the inhibitor, i KK R ; = $(+g + $) + [++-(l 52 +& (91 1 i 1 :B When glucose 6-phosphate was the variable substrate and glucosamine 6-phosphate the inhibitor, Equation 4 was arranged in the following manner:.52 b K ; = ~L~+-( +~ll + (&*+ (10) 1 1 1 For an ordered addition of Substrates A and B, inhibition by an analogue of B forming an EA dead-end complex will be uncompetitive with respect to A as varied substrate, as predicted by Equation 9. Fig. 6 shows the results of this experiment in which the concentration of NADP+ was varied at different glucosamine B-phosphate concentrations with the glucose 6-phosphate concentration maintained at 130 /LM. The pattern of inhibition exhibited by glucosamine 6-phosphate with respect to NADP+ is uncompetitive. A replot of the intercepts versus glucosamine B-phosphate is given in Fig. 7 and is linear up to an inhibitor concentration of 10 mm at which point an unknown factor gives rise to a parabolic effect. The K, for the E.NADP+.Z complex is 5.1 mm. As predicted by Equation 10, glucosamine 6-phosphate exhibits competitive inhibition with respect to glucose 6.phosphate as is shown in Fig. 8. The inhibition constant, K,, when calculated from the replot of slopes of Fig. 8 versus inhibitor concentration is 4.4 mm which is in excellent agreement with the K, value determined with NADP+ as the variable substrate. nhibition by ADP-The kinetics of ADP inhibition have been done on glucose-6-phosphate dehydrogenase from human platelets (4), however, there is no discussion as to what enzyme form the ADP binds with. The data and inhibition plots presented do not conform with the inhibition patterns to be expected with equations using Cleland s nomenclature (6, 8). The equation used here for the ADP inhibition studies when NADP+ is the variable substrate, is a limited version of Equation 4 in the form: When glucose 6-phosphate was the variable substrate, Equation 11 was rewritten in the following manner: The data were consistent with the above equations. Fig. 9 indicates that ADP inhibition was competitive with respect to NADP+ and the inhibition constant obtained from the replot of inhibitor (ADP) concentrations versus slopes of Fig. 9 was 2.64 rnm. When glucose B-phosphate was the variable substrate versus different inhibitor concentrations, noncompetitive inhibition was observed in Fig. 10. The inhibition constants when calculated from the replot of intercepts and slopes of Fig. 10 uersus inhibitor concentration are 1.43 and 1.66 mm. The average of the three K, values is 1.91 mm. ATP nhibition-the kinetics of ATP inhibition have been done on the enzyme from human platelets (4). At ATP levels below 2 mm the inhibitor combines primarily with the EA form of the enzyme. At higher levels of ATP, the inhibitor also competes with NADP+ for the free form (E). n this study, the equation applied when NADP+ is the variable substrate uersus ATP is a rearrangement of Equation 4 in the form: When glucose 6.phosphate was the variable substrate versus ATP, Equation 4 was used in the following form: The results were consistent with the above two equations. ATP inhibition is noncompetitive with respect to both NADP+ and glucose 6-phosphate as is given in Figs. 11 and 12. Thus ATP seems to combine with both the E and EA forms of the enzyme. Fig. 13 contains a replot of the slopes and intercepts from Fig. 11 which are linear up to 3 mm, at which point a parabolic effect is visible. Using the linear portions of these plots, values of 7.2 mm and 4.5 mm were calculated for K, and K,. The same constants determined from the replots in Fig. 14. FG. 6. Glucosamine 6.phosphate (Glu-6-P) inhibition plot at 30". Glucose &phosphate concentration is 130 PM, NADP+ concentration varied from 7.5 to 25 pm, and glucosamine 6.phosphate concentration varied from 4 to 12 mm, E, = 0.0081 PM. FG. 8. Glucosamine 6.phosphate (GlcN-6-P) inhibition plot with glucose 6.phosphate (Glc-6-P) as the variable substrate. NADP concentration is 25 PM, glucose 6.phosphate concentration varied from 30 to 130 JLM and glucosamine &phosphate concentration varied from 4 to 10mM. E, = 0.0081~~.

Glucose-6-P Dehydrogenase Kinetics 2261 0.O.10.5 pml.1 FG. 9. ADP inhibition with NADP- as the variable substrate. Glucose 6.phosphate concentration is 130 @, NADP+ concentration varied from 7.5 to 50 PM and ADP concentration varied from 1 to 3 mu. E, = 0.0081 KM. ooo.05.10 FG. 11. ATP inhibition with NADP as the variable substrate. Glucose 6.phosphate concentration is 130 PM, NADP+ concentration varied from 6.25 to 50 PM and ATP concentration varied from 2 to 5 mm. E, = 0.0081 um. rm-l. 5 FG. 10. ADP inhibition with glucose 6.phosphate (Glc-6-P) as the variable substrate. NADP concentration is 25 pm, glucose 6-phosphate concentration varied from 50 to 160 KM and ADP concentration varied from 1 to 3 mm. E, = 0.0081 PM. were 6.7 mm and 6.0 mm, respectively. Taking an average of these values results in an E.ATP dissociation constant of 7 mm and E.NADP+.ATP dissociation constant of 5.2 mm, respectively. DSCUSSON n this work, initial velocity experiments on pig liver glucose-6-phosphate dehydrogenase indicate that the mechanism is sequential, thus excluding a ping-pong mechanism. The combination of results from initial velocity and product inhibition patterns are consistent with an ordered addition of substrates with NADP+ being the first substrate. Results from dead-end inhibition studies using an analogue of glucose 6- phosphate, help to rule out a rapid equilibrium random with a dead-end complex mechanism. n a random mechanism, glucosamine 6-phosphate can be expected to combine with both the free and E.NADP+ enzyme forms. This would result in noncompetitive inhibition with respect to NADP+ and competitive inhibition with respect to glucose 6-phosphate. However, the inhibition patterns observed were uncompetitive with respect to NADP+ and competitive with respect to glucose 6-phosphate, which indicates that the inhibitor binds to the E.NADP+ form of the enzyme. Since the inhibitor is an analogue of glucose 6-phosphate, one may conclude that glucose 6-phosphate also binds to the E.NADP+ form of the enzyme, and is the second substrate in the reaction mechanism. Elimination of the Theorell-Chance mechanism from further consideration can be done by the demonstration of significant central complex concentrations. This can be accomplished by dead-end inhibition studies using an inhibitor shown to com- loo0 /&&M hl-.o FG. 12. ATP inhibition with glucose 6.phosphate (Glc-6-P) as the variable substrate. NADP+ concentration is 25 KM, glucose 6-phosphate concentration varied from 80 to 400 FM, and ATP concentration, varied from 2 to 5 mm. E, = 0.0081 PM. bine only with the central complexes. Unfortunately, such an inhibitor has not been found for the glucose-6-phosphate dehydrogenase reaction. Product inhibition studies using the first product might also rule out a Theorell-Chance mechanism, however, 6.phosphogluconolactone is highly unstable in aqueous solution (half-life 1.5 s) (13). n summary, results from initial velocity studies, product inhibition, and dead-end inhibition kinetics are consistent with an ordered mechanism in which coenzyme adds first and is released last from pig liver glucose-6-phosphate dehydrogenase. 1. 2. 3. 4. 5. 6. 7. 8. 9. 10. 11. 12. 13. 14. 15. REFERENCES Alfolyan, A., and Luzzatlo, A. (1971) Biochemistry 10, 415-419 Soldin, S. J., and Balinsky, D. (1968) Biochemistry 7, 1077-1082 Olive, C., Geroch, M. E., and Levy, H. R. (1971) J. Biol. Chem. 246, 2047-2057 Kosow, D. P. (1974) Arch. Biochem. Biophys. 162, 186193 Kanji, M.., Toews, M. L., and Carper, W. R. (1976) J. Biol. Chem. 251,2255-2257 Cleland, W. W. (1963) Biochim. Biophys. Acta 67,104-137 King, E. L., and Altman, C. (1956) J. Phys. Chen. 60,1375-1378 Cleland, W. W. (1963) Biochim. Biophys. Acta 67, 173-187 Lineweaver, H., and Burk, D. (1934) J. Am. Chem. Sot. 56, 658-666 Cleland, W. W. (1963) Nature 198, 4633465 Carper, W. R., Chang, K. W., Thorpe, W. G., Carper, M. A., and Buess, C. M. (1974) Biochim. Biophys. Acta 358, 49-56 UPAC-UB Commission on Biochemical Nomenclature (1966) J. Biol. Chem. 241. 527-533 Horecker, B. L., and Smyrniotis, P. Z. (1953) Biochim. Biophys. Acta 12. 98-102 Fromm, H. J. (1964) Biochim. Biophys. Acta 81, 413-417 Bessell, E. M., and Thomas, P. (1973) Biochem. J. 131, 83-89

2262 Glucose-6-P Dehydrogenase Kinetics