A STUDY OF STRUCTURE AND FUNCTION RELATIONSHIPS IN TRANSHYDROGENASE FROM PSEUDOMONAS AERUGINOSA

Pseudomonas aeruginosa transhydrogenase (PATH) catalyzes the transfer of hydrogen from NADPH to NAD('+) under standard reaction conditions. The addition of 2'AMP or Ca('++) to the assay system enhances the reactivity toward NADH.;Immobilization of PATH upon various supports by covalent and absorptive methods was performed. When bound to silica glass, the coupled enzyme showed full activation of the NADH reaction without prior exposure to 2'AMP or Ca('++). The K(,NADPH) for this system was calculated to be 1.31 x 10('-1) mM. The K(,NADH) for the same system was 2.5 x 10('-1) mM. Other immobilized species gave values within a 0.1 mM range of the silica glass. The kinetics of the immobilized PATH was found to be first order with respect to NADPH and NADH. The bound enzyme showed cooperativity in the binding of NADPH, while NADH appeared to act as a substrate inhibitor. NADP('+) was found to be a mixed-type inhibitor in accordance with the ping pong bi bi or Theorell-Chance mechanisms.;Stabilty and pH studies showed little change in the characteristics of the enzyme after immobilization. Heat denatured immobilized PATH was partially reactivated by FAD. Urea denatured bound PATH was not reactivated by the addition of FAD. The immobilized PATH was slightly more susceptible to trypsin digestion than the soluble enzyme.;Lysine, histidine, tyrosine and arginine residues in soluble PATH were selectively modified by trinitrobenzenesulfonic acid, rose bengal, trinitromethane, and 1,2-cyclohexanedione, respectively. Samples treated with the two former reagents gave products with enhanced NADH activity without 2'AMP. Treatment with the two latter reagents did not produce an increase in the rate of the NADH reaction. Active enzyme ultracentrifugation indicated that the active modified structures were smaller than the 34 S forms associated with PATH, PATH/2'AMP, and PATH/Ca('++), with active sedimentation values ranging from 23.0-26.5 S.;PATH was purified via affinity chromatography with a N('6)-hexyl-2',5'-ADP-Sepharose 4B column. The enzyme was purified 276-fold by the affinity chromatography and was 576-fold purified after subsequent chromatography upon a Bio-Gel A-0.5 column. The protein was estimated to be over 90% pure by SDS gel electrophoresis. N('8)-hexyl-2',5'-ADP was tried as an affinity ligand, but complete elution from this column could not be effected. Purified PATH was found to be stabilized by 20% glycerol at -20(DEGREES) C.;Antibodies were made against the native PATH. In an Ouchterlony double diffusion gel against the crude sera, PATH activated with 2'AMP and Ca('++) gave lines of complete identity with the native PATH. A time course of activity retained by PATH during incubation with anti-PATH was also made. An initial increase in NADH activity was observed. It was followed by a rapid reduction in the activity of the NADH reaction. The NADPH and NADH/2'AMP reactions showed a consistent decrease in activity throughout the timed reaction.;It was concluded that the regulatory region of PATH is more vulnerable than the active site to solvents and reagents. It was also concluded that 2'AMP and Ca('++) exert their effect by reducing the flexibility of the enzyme.