Kinetics and Equilibria of the Reductive and Oxidative Half-Reactions of Human Renalase with α-NADPH

Renalase is a recently discovered flavoprotein that has been reported to be a hormone produced by the kidney to down-modulate blood pressure and heart rate. The consensus belief has been that renalase oxidizes circulating catecholamine neurotransmitters thereby attenuating vascular tone. However, a convincing in vitro demonstration of this activity has not been made. We have recently discovered that renalase has alpha-NAD(P)H oxidase/anomerase activity. Unlike most naturally occurring nucleotides, NAD(P)H can accumulate small amounts of the alpha-anomers that once oxidized are configurationally stable and unable to participate in cellular activity. Thus, anomerization of NAD(P)H would result in a continual loss of cellular redox currency. As such, it appears that the root purpose of renalase is to return alpha-anomers of nicotinamide dinucleotides to the beta-anomer pool. In this article, we measure the kinetics and equilibria of renalase in turnover with alpha-NADPH. Renalase is selective for the alpha-anomer, which binds with a dissociation constant of similar to 20 +/- 3 mu M. This association precedes monophasic two-electron reduction of the FAD cofactor with a rate constant of 40.2 +/- 1.3 s(-1). The reduced enzyme then delivers both electrons to dioxygen in a second-order reaction with a rate constant of similar to 2900 M-1 s(-1). Renalase has modest affinity for its beta-NADP(+) product (K-d = 2.2 mM), and the FAD cofactor has a reduction potential of -155 mV that is unaltered by saturating beta-NADP(+). Together these data suggest that the products are formed and released in a kinetically ordered sequence (beta-NADP(+) then H2O2), however, the reoxidation of renalase is not contingent on the dissociation of beta-NADP(+). Neither the oxidized nor the reduced form of renalase is able to catalyze anomerization, implying that the redox and anomerization chemistries are inextricably linked through a common intermediate.