ASSIMILATORY NITRATE REDUCTASE - REDUCTION AND INHIBITION BY NADH NAD(+) ANALOGS/

Assimilatory nitrate reductase from Chlorella vulgaris catalyzes the r ate-limiting step, the conversion of nitrate to nitrite, in nitrate as similation. Initial rate studies of nitrate reductase activity, perfor med under optimum conditions of constant ionic strength (mu = 0.2) and pH (8.0) and using NADH as reductant, indicated the absence of substr ate inhibition at NADH concentrations below 300 mu M and NO3- concentr ations less than 3 mM. Chlorella nitrate reductase exhibited a marked preference for NADH (V-max = 9.2 mu mol NADH/min/nmol heme and K-m = 2 .3 mu M) as the physiological electron donor but could also utilize al pha-NADH (V-max = 5.6 mu mol NADH/min/nmol heme and K-m = 131 mu M) an d NADPH (V-max = 0.6 mu mol NADPH/min/nmol heme and K-m = 910 mu M) th ough with significantly decreased efficiency. Examination of various N ADH-analogs indicated that reduced nicotinamide hypoxanthine dinucleot ide (NHDH) was used most efficiently (V-max = 9.3 mu mol NHDH/min/nmol heme and K-m = 7.9 mu M), while reduced nicotinamide mononucleotide ( NMNH) was utilized least efficiently (V-max = 0.07 mu mol NMNH/min/nmo l heme and K-m = 676 mu M). Overall, modifications to the nicotinamide moiety or the addition of a phosphate group were observed to result i n the most significant decreases in V-max, indicating poor reducing su bstrates. Product inhibition studies indicated both NAD(+) (K-i = 2.2 mM) and NADP(+) (K-i = 10.5 mM) to be competitive inhibitors of Chlore lla NR. A variety of NAD(+) analogs were also determined to act as com petitive inhibitors with varying degrees of efficiency. 3-Pyridinealde hyde adenine dinucleotide was the most efficient inhibitor (K-i = 0.74 mM) while nicotinamide was the least efficient (K-i = 18.1 mM). Overa ll, changing substituents on the nicotinamide ring or its complete del etion produced the most effective inhibitors compared to NAD(+). In co ntrast, changes in the adenine or ribose moieties produced less effect ive inhibitors when compared to NAD(+). These results represent the mo st comprehensive analysis of the effect of modifications of the physio logical reductant (NADH) and product (NAD(+)) on nitrate reductase act ivity. (C) 1994 Academic Press, Inc.