G6PDH

Pentose phosphate pathway (= hexose monophosphate shunt) generation of NADPH G6PDH deficiency... 

Immobilization of the enzymes GR and G6PDH was carried out by coupling them to Afh-Gel 10 support. Affi-Gel 10 is an A-hydroxysuccinimide ester of a derivatized cross-linked agarose gel. Upon addition of the enzyme to Affi-Gel 10, the A-hydroxysuccinimide is displaced and the free amino groups of the enzyme form a stable amide bond with the gel. The enzymes GR and G6PDH were immobilized separately following the procedure described below. 

All the solutions were prepared in 0.1 M Tris buffer pH 8 containing 10 mM MgCl2 and 0.94 mM EDTA. The increase in absorption at 339 nm was measured for 5 min, and a unit of G6PDH is defined as the amount of enzyme that will yield 1 /rmol of NADPH per minute. 

Kinetic Parameters and Mechanism of a Reaction Catalyzed by G6PDH... 

From Figure 4.54 it can he seen that the family of reciprocal plots obtained at different fixed concentrations of NADP are essentially parallel to one another. This is also indicated in Figure 4.55, where the value of the slopes of the tines seem to be approximately constant. These results imply that the velocity equation for the ping-pong mechanism [146] can be used to describe the rate of the reaction catalyzed by G6PDH. Although initial velocity studies alone cannot define the exact kinetic mechanism [146,147], we are more interested in the appropriate rate equation that describes the reaction progress. 

Figure 4.59 Experimental and theoretical results obtained for the basic system using G6PDH and GR, and operated as a fed-batch reactor. Results were obtained with Vm.oePDH = =...

Figure 4.59 presents the results obtained when the basic system, containing G6PDH and GR, was operated as a fed-batch reactor in the configuration described in Figure 4.58. For comparison, the results of pertinent numerical simulations are also shown. It can be seen that the signal obtained in the experimental system indeed follows the characteristic course shown by the signal calculated, but the actual numerical values are different. This dissimilarity has been attributed to inhibition effects in the reactions involved, effects that were not considered in the calculations. Therefore, a search for potential inhibitors was undertaken.

Based on the results of the preceding section, potential inhibitors to the enzymes involved, G6PDH and GR, were searched for among the compounds that participate in the experimental system considered. It was found that GR is inhibited by G6P, the substrate of the other reaction. To determine the inhibition constant, G6P was considered as a dead-end inhibitor [146] that... 

Kj.gr = 0.136 mM/min. In Figure 4.66 theoretical results obtained from the analyhcal model without considering inhibihon of G6PDH by NADPH are also presented. It can be seen that when inhibihon by G6P was not considered in the analyhcal model (A g6p = oo), the theorehcal results are very different from the experimental results. This agreement improves when inhibihon... 

Figure 4.66 Experimental and theoretical results obtained for the basic system using G6PDH and GR and operated as a fed-batch reactor. Theoretical results were obtained with Fm,G6PDH = 0.211 mM/min and Um.GR = 0.136 mM/min and considering G6P as an inhibitor to GR with the indicated values of Ki Qgp (oo = no inhibition by G6P). No inhibition of G6PDH by NADPH is considered here.

Table 4.10 Results Obtained in Inunobihzation of G6PDH and GR on Affi-Gel 10...

The interpretation of the experimental results presented in Figure 4.69 was extended to include inhibition of the enzyme G6PDH by NADPH with i.NADPH = 0.027 mM. A comparison between experimental and calculated results is shown in Figure 4.70. In this case better agreement is achieved when lower values of Ki oee are employed, the values being in the range obtained in experiments carried out in a fed-batch reactor (0.15 to 1 mM). 

Figure 4.70 Experimental and theoretical results obtained for the basic system with G6PDH and GR when operated as a packed bed reactor. Theoretical results were obtained with n = 5 (i.e., assuming a plug flow regimen), Vm.oePDH = 0.6 mM/min, and = 0.7 mM/min...

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