6-Phosphogluconate Dehydrogenase PURIFICATION AND KINETICS

A method is described for the isolation and purification of 6-phosphogluconate dehydrogenase from pig liver. The molecular weight is estimated at 83,000 and that of the subunits is 42,000 as determined by gel electrophoresis. The pH maximum is 8.5 in 50 mM glycine/NaOH buffer and from 7.5 to 10 in 50 mM phosphate buffer at 30”. Magnesium ion is not required for activity and acts as an inhibitor at concentrations above 20 mM. A cellular fractionation study indicates that this enzyme is located almost entirely within the soluble portion of the cytoplasm. Kinetic studies have been done in 50 mM glycine buffer, pH 8.5, at 30”. The data are consistent with a sequential mechanism in which NADP+ is added first, followed by 6-phosphogluconate, and the products are released in the order, COz, ribulose 5-phosphate, and NADPH. The Michaelis constant is 13.5 PM for 6-phosphogluconate. Dissociation constants are 4.8 FM for NADP+ and 5.1 PM for NADPH.

A method is described for the isolation and purification of 6-phosphogluconate dehydrogenase from pig liver. The molecular weight is estimated at 83,000 and that of the subunits is 42,000 as determined by gel electrophoresis.
The pH maximum is 8.5 in 50 mM glycine/NaOH buffer and from 7.5 to 10 in 50 mM phosphate buffer at 30". Magnesium ion is not required for activity and acts as an inhibitor at concentrations above 20 mM. A cellular fractionation study indicates that this enzyme is located almost entirely within the soluble portion of the cytoplasm.
Kinetic studies have been done in 50 mM glycine buffer, pH 8.5, at 30". The data are consistent with a sequential mechanism in which NADP+ is added first, followed by 6-phosphogluconate, and the products are released in the order, COz, ribulose 5-phosphate, and NADPH. The Michaelis constant is 13 catalyzes the reversible oxidative decarboxylation of 6-phospho-D-gluconate to yield Dribulose 5-phosphate and COB, with NADP+ being reduced to NADPH (Fig. 1). In previous studies (1,2) we have reported the isolation, Lharacterization, and steady state kinetics of glucose-6-phosphate dehydrogenase from pig liver. In this work, pig liver 6phosphogluconate dehydrogenase has been isolated, purified, characterized, and its forward reaction kinetics has been investigated. 6-Phosphogluconate dehydrogenase has been isolated from numerous nonmammalian sources , and was partially purified from rat liver by Glock and McLean in 1953 (8). Other isolation and purification studies from mammalian sources include sheep liver (g-11), rat liver (12), and human erythrocytes (13).
The kinetics of the oxidative decarboxylation reaction catalyzed by the enzyme from Can&da utilis was studied in 1961, and a random order mechanism was postulated (4). Michaelis constants for NADP+ and 6-phosphogluconate have been reported for the enzyme from several sources (6,7,14 The 15min fractions of approximately 3 ml each were collected.
The most active fractions eluted from this column were combined and lyophilized to dryness. The resulting powder was redissolved in 5 ml of 0.2 M phosphate buffer, pH 7.0, and again passed down the same Sephadex G-200 column under the same conditions.
The most active fractions from the second Sephadex G-200 run (5 to 10 ml) were added to a Cellex P column equilibrated with 0.005 M phosphate buffer, pH 6.0, and the column was washed with about 10 ml of the same buffer. The protein was then eluted from the column by washing with successively higher concentrations of buffer, a stepwise gradient from 0.005 to 0.2 M being created by adding 0.005 M, 0.01 M, 0.05 M, and finally 0.2 M phosphate buffers at pH 6.0. The flow rate was about 0.6 ml/min, with 5-min fractions of 3 ml each being collected.
The enzyme eluted from the Cellex P column was stored in solution at 4".
Although an accurate assay of the homogenate is not possible due to the turbidity of the solution, the acid treatment gives a 2-to 5-fold increase in specific activity with very little loss of total activity. Extracts not acid-treated remain turbid even after the ammonium sulfate precipitate is redissolved, and separation on Sephadex G-200 is much poorer. The specific activity of the final Cellex P sample is 0.4 unitlmg.
A typical set of results is given in Table I  above mechanisms, and any deviations were then accounted for by alterations of this mechanism. In all equations used in this work, v is the measured initial velocity, V, is the maximum velocity for the forward reaction, and K,, is the equilibrium constant; A, B, P, Q, and R are concentrations, K,, Kb, KS, K,, and K, are Michaelis constants, and Ki,, Kib, KiP, KiP, and Ki, are dissociation constants for NADP+, GlcA-GP,' CO%, Rbu-5-P, and NADPH, respectively.
The steady state rate equation for the mechanism in Equation 1 can be derived by analysis of the King-Altman  concentration of the variable substrate will yield a family of intersecting straight lines whose slopes and intercepts are a function of the reciprocal concentration of the nonvaried substrate.
Cleland (21) described how product inhibition studies can be used to verify enzyme kinetic mechanism and help establish the order of substrate binding and product release. Initial velocity equations for product inhibition studies are Equations 5 through 10 (see below).

Forward
Reaction-For studies of the forward reaction, initial velocity measurements were made at six concentrations of GlcA-6-P for each of six different concentrations of NADP+. The data were computer fitted to Equation 2. A plot of reciprocal velocity versus reciprocal concentration of GlcA-6-P (Fig.  2) gives a series of straight lines which intersect at a common point as predicted by Equation 2. This result shows that the Both of these equations are linear equations, and a Line-mechanism is a sequential one in which all substrates are weaver-Burk (33) plot of reciprocal velocity versus reciprocal bound before any products are released. The Michaelis constants are 13.5 (kO.5) pM for NADP+ (K,) and 29.2 (k1.0) pM ' The abbreviations and symbols used are those recommended in for GlcA-6-P (K,). The dissociation constant for the E. NADP+ NADPH inhibition with GlcA-6-P as the variable substrate at 30". NADP is 50 FM, GlcA-6-P varied from 25 to 500 PM, and NADPH varied from 6.25 to 25 PM.
V,, was 0.00493 (r0.00006) pM/min. These values are in the same range as those determined for the sheep liver enzyme (15). NADPH Inhibition-For NADPH product inhibition studies with NADP' as the variable substrate, the GlcA-6-P concentration was held constant at 0.50 mM. The NADP+ concentration was varied between 10 and 100 PM for each of three levels of NADPH. The results fit Equation 9 which predicts the linear competitive inhibition shown in Fig. 3. Ki, was calculated from the slope of a linear slope versus NADPH concentration replot (not shown) and is 5.6 PM.
For NADPH product inhibition studies where GlcA-6-P was the variable substrate, the NADP+ concentration was held constant at 50 pM. A plot of the data is shown in Fig. 4 and the inhibition is noncompetitive as predicted by Equation 10. The slope and intercept replots are linear as predicted and the value for K,, from the slope replot is 4.0 PM and that from the intercept replot is 5. gluconate dehydrogenase would therefore be controlled by the relative abundance of NADP+ and NADPH present in the cell.
Ribulose 5-Phosphate Inhibition -Product inhibition studies using ribulose 5-phosphate as inhibitor and NADP+ as the variable substrate were run at a constant GlcA-6-P concentration of 0.50 mM. A plot of the results is shown in Fig. 5, and the inhibition pattern is uncompetitive, as predicted by Equation 7.
For ribulose 5-phosphate inhibition studies with GlcA-6-P as the variable substrate, the NADP+ concentration was held constant at 50 to 100 PM at ribulose 5-phosphate concentrations of 70, 140, and 280 PM. Fig. 6  CO, Inhibition-Results of studies of CO, inhibition on this enzyme give the inhibition patterns predicted by Equations 5 and 6 for simple product inhibition. The inhibition is noncompetitive; however, the slope and intercept replots are parabolic at CO, concentrations above 1.0 mM. This suggests that CO, combined with some form(s) of the enzyme in dead-end fashion in addition to its product inhibition due to combination with the E&R form.
For studies with NADP+ as the variable substrate, the GlcA-6-P concentration was held constant at 65 PM, NADP+ concentration was varied from 10 to 50 pM, COZ concentrations were in the range from 0.56 to 1.39 mM as is shown in Fig. 7. When GlcA-6-P was the variable substrate, the NADP+ concentration was 50 PM, GlcA-6-P concentrations were varied from 0.1 to 0.65 mM, and CO, concentrations were varied from 0.56 to 1.39 mM. Values of 0.0169 and 0.0134 PM-' were obtained for K,/K, Ki, from the slope of the slope replots of these experiments as is shown in Fig. 8. Dead-end inhibition by CO, has not been reported previously for 6-phosphogluconate dehydrogenase, however, dead-end inhibition of CO, with the free enzyme has been reported for the oxidative decarboxylation reactions catalyzed by malic enzyme (30). CONCLUSION All of the results obtained in the kinetic studies are consistent with the proposed Ordered Bi-Ter mechanism. The inhibi- inhibition. This latter case is observed both for this enzyme and for the sheep liver enzyme (15).