Effect of salt and pH on the reductive half-reaction of Mycobacterium tuberculosis FprA with NADPH.
Despite a number of studies, the formation of the Michaelis complexes between ferredoxin-NADP (+) reductases and NADP(H) eluded detailed investigations by rapid kinetic techniques because of their high formation rates. Moreover, the reversible nature of the reaction of hydride ion transfer between these enzymes and NADPH prevented the obtainment of reliable estimates of the rate constant of the hydride transfer step. Here we show that by working at a high salt concentration, the mechanism of the reaction with NADPH of FprA, a Mycobacterium tuberculosis homologue of adrenodoxin reductase, is greatly simplified, making it amenable to investigation by rapid reaction techniques. The approach presented herein allowed for the first time the observation of the formation of the Michaelis complex between an adrenodoxin reductase-like enzyme and NADPH, and the determination of the related rate constants for association and dissociation. Furthermore, the rate constant for the reaction of hydride ion transfer between NADPH and FAD could be unambiguously assessed. It is proposed that the approach described should be applicable to other ferredoxin reductase enzymes, providing a valuable experimental tool for the study of their kinetic properties.
Pennati
Andrea
A
Dipartimento di Scienze Biomolecolari e Biotecnologie, Università degli Studi di Milano, via Celoria 26, 20133 Milano, Italy.
Zanetti
Giuliana
G
Aliverti
Alessandro
A
Gadda
Giovanni
G
D000693:Anaerobiosis; D005287:Ferredoxin-NADP Reductase / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009169:Mycobacterium tuberculosis / Q000201:enzymology*; D009247:NADH, NADPH Oxidoreductases / Q000737:chemistry / Q000378:metabolism*; D009249:NADP / Q000737:chemistry / Q000378:metabolism*; D010084:Oxidation-Reduction; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D012965:Sodium Chloride / Q000737:chemistry*; D013056:Spectrophotometry, Ultraviolet
D011994:Recombinant Proteins; D012965:Sodium Chloride; D009249:NADP; C464438:FprA protein, M tuberculosis; D005287:Ferredoxin-NADP Reductase; D009247:NADH, NADPH Oxidoreductases