Catalytic mechanism and substrate specificity of the beta-subunit of the voltage-gated potassium channel.
The beta-subunits of voltage-gated potassium (Kv) channels are members of the aldo-keto reductase (AKR) superfamily. These proteins regulate inactivation and membrane localization of Kv1 and Kv4 channels. The Kvbeta proteins bind to pyridine nucleotides with high affinity; however, their catalytic properties remain unclear. Here we report that recombinant rat Kvbeta2 catalyzes the reduction of a wide range of aldehydes and ketones. The rate of catalysis was slower (0.06-0.2 min(-1)) than those of most other AKRs but displayed the expected hyperbolic dependence on substrate concentration, with no evidence of allosteric cooperativity. Catalysis was prevented by site-directed substitution of Tyr-90 with phenylalanine, indicating that the acid-base catalytic residue, identified in other AKRs, has a conserved function in Kvbeta2. The protein catalyzed the reduction of a broad range of carbonyls, including aromatic carbonyls, electrophilic aldehydes and prostaglandins, phospholipids, and sugar aldehydes. Little or no activity was detected with carbonyl steroids. Initial velocity profiles were consistent with an ordered bi-bi rapid equilibrium mechanism in which NADPH binding precedes carbonyl binding. Significant primary kinetic isotope effects (2.0-3.1) were observed under single- and multiple-turnover conditions, indicating that the bond-breaking chemical step is rate-limiting. Structure-activity relationships with a series of para-substituted benzaldehydes indicated that the electronic interactions predominate during substrate binding and that no significant charge develops during the transition state. These data strengthen the view that Kvbeta proteins are catalytically active AKRs that impart redox sensitivity to Kv channels.
Tipparaju
Srinivas M
SM
Division of Cardiology, Department of Medicine, Institute of Molecular Cardiology, University of Louisville, Kentucky 40202, USA.
Barski
Oleg A
OA
Srivastava
Sanjay
S
Bhatnagar
Aruni
A
D000429:Alcohol Oxidoreductases / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D000449:Aldehyde Reductase; D000447:Aldehydes / Q000378:metabolism; D000074408:Aldo-Keto Reductases; D000818:Animals; D001665:Binding Sites; D002384:Catalysis; D003903:Deuterium; D008401:Gas Chromatography-Mass Spectrometry; D006863:Hydrogen-Ion Concentration; D007659:Ketones / Q000378:metabolism; D007700:Kinetics; D016297:Mutagenesis, Site-Directed; D009154:Mutation; D009249:NADP / Q000378:metabolism; D010743:Phospholipids / Q000378:metabolism; D024642:Potassium Channels, Voltage-Gated / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D011453:Prostaglandins / Q000378:metabolism; D021122:Protein Subunits / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization; D013379:Substrate Specificity
D000447:Aldehydes; D007659:Ketones; D010743:Phospholipids; D024642:Potassium Channels, Voltage-Gated; D011453:Prostaglandins; D021122:Protein Subunits; D011994:Recombinant Proteins; D009249:NADP; D003903:Deuterium; D000429:Alcohol Oxidoreductases; D000074408:Aldo-Keto Reductases; D000449:Aldehyde Reductase