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 ß-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 ß-NADP+ product (Kd=2.2 mM), and the FAD cofactor has a reduction potential of -155 mV that is unaltered by saturating ß-NADP+. Together these data suggest that the products are formed and released in a kinetically ordered sequence (ß-NADP+ then H2O2), however, the reoxidation of renalase is not contingent on the dissociation of ß-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.

Renalase is an α-NAD(P)H oxidase/anomerase.

Renalase is a protein hormone secreted into the blood by the kidney that is reported to lower blood pressure and slow heart rate. Since its discovery in 2005, renalase has been the subject of conjecture pertaining to its catalytic function. While it has been widely reported that renalase is the third monoamine oxidase (monoamine oxidase C) that oxidizes circulating catecholamines such as epinephrine, there has been no convincing demonstration of this catalysis in vitro. Renalase is a flavoprotein whose structural topology is similar to known oxidases, lysine demethylases, and monooxygenases, but its active site bears no resemblance to that of any known flavoprotein. We have identified the catalytic activity of renalase as an α-NAD(P)H oxidase/anomerase, whereby low equilibrium concentrations of the α-anomer of NADPH and NADH initiate rapid reduction of the renalase flavin cofactor. The reduced cofactor then reacts with dioxygen to form hydrogen peroxide and releases nicotinamide dinucleotide product in the ß-form. These processes yield an apparent turnover number (0.5 s(-1) in atmospheric dioxygen) that is at least 2 orders of magnitude more rapid than any reported activity with catechol neurotransmitters. This highly novel activity is the first demonstration of a role for naturally occurring α-NAD(P)H anomers in mammalian physiology and the first report of a flavoprotein catalyzing an epimerization reaction.