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 α-NAD(P)H oxidase/anomerase activity. Unlike most naturally occurring nucleotides, NAD(P)H can accumulate small amounts of the α-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 α-anomers of nicotinamide dinucleotides to the β-anomer pool. In this article, we measure the kinetics and equilibria of renalase in turnover with α-NADPH. Renalase is selective for the α-anomer, which binds with a dissociation constant of ∼20±3 μM. 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 ∼2900 M(-1) s(-1). Renalase has modest affinity for its β-NADP+ product (Kd=2.2 mM), and the FAD cofactor has a reduction potential of -155 mV that is unaltered by saturating β-NADP+. Together these data suggest that the products are formed and released in a kinetically ordered sequence (β-NADP+ then H2O2), however, the reoxidation of renalase is not contingent on the dissociation of β-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.