Kinetic Characteristics of the Pyridine Nucleotide Transhydrogenase from Pseudomonas aeruginosa

Abstract

Abstract Kinetic studies on Pseudomonas aeruginosa pyridine nucleotide transhydrogenase yield the following information. In the presence of 5 x 10-4 m 2'-AMP the initial velocity experiments for both the TPNH-(TN)DPN+ (oxidized thionicotinamide DPN) and DPNH-(TN)DPN+ reactions are consistent with a bi bi catalytic reaction mechanism. DPN+ inhibition of the DPNH-(TN)DPN+ reaction is the type expected for this mechanism. When 2'-AMP is not included in the TPNH-(TN)DPN+ system the reaction velocity shows a second order dependence on TPNH, but a first order dependence on (TN)DPN+. The maximum velocity for the TPNH-(TN)DPN+ system is apparently the same in the presence as in the absence of 5 x 10-4 m 2'-AMP. However, in the presence of 5 x 10-4 m 2'-AMP the reaction velocity shows a first order dependence on both substrates. These experiments show that both TPNH and 2'-AMP are activators of the enzyme. In the DPNH-(TN)DPN+ reaction the velocity shows a first order dependence on both substrates and a second order dependence on 2'-AMP. 2'-AMP causes a decrease in the apparent Michaelis constant for DPNH and an increase in the apparent maximum velocity. If the mechanism is assumed to be ping-pong bi bi then the experiments indicate that 2'-AMP increases the rates of steps in the portion of the reaction sequence involving reduction of the enzyme by DPNH. Experiments indicate that TPN+ does not have the same capacity for activation as do TPNH and 2'-AMP. In contrast to DPN+, TPN+ at low concentrations is competitive with DPNH in the DPNH-(TN)DPN+ reaction. Thus failure of the transhydrogenase to catalyze the reaction between DPNH and TPN+ can be explained by the failure of TPN+ to cause full activation, making DPNH a less effective substrate, and also by formation by TPN+ of a dead end complex with enzyme.