Studies on the active site of the Neurospora crassa plasma membrane H+-ATPase with periodate-oxidized nucleotides.
The Neurospora crassa plasma membrane H+-ATPase is inactivated by the periodate-oxidized nucleotides, oATP, oADP, and oAMP, with oAMP the most effective. Inhibition of the ATPase is essentially irreversible, because Sephadex G-50 column chromatography of the oAMP-treated ATPase does not result in a reversal of the inhibition. Inhibition of the ATPase by oAMP is protected against by the H+-ATPase substrate ATP, the product ADP, and the competitive inhibitors TNP (2',3'-O-(2,4,6-trinitrocyclohexadienylidine)-ATP and TNP-ADP, suggesting that oAMP inhibition occurs at the nucleotide binding site of the enzyme. The rate of inactivation of the ATPase by oAMP is only slightly affected by EDTA, indicating that the oAMP interaction with the nucleotide binding site of the H+-ATPase occurs in the absence of a divalent cation. The protection against oAMP inhibition by ADP is likewise unaffected by EDTA. The inhibition of the ATPase by oAMP is absolutely dependent on the presence of acidic phospholipids or acidic lysophospholipids known to be required for H+-ATPase activity, suggesting that these lipids either aid in the formation of the nucleotide binding site or render it accessible. Incubation of the ATPase with Mg2+ plus vanadate, which locks the enzyme in a conformation resembling the transition state of the enzyme dephosphorylation reaction, completely protects against inhibition by oAMP, suggesting that in this transition state conformation the nucleotide site either does not exist, or is inaccessible to oAMP. Labeling studies with [14C] oAMP indicate that the incorporation of 1 mol of oAMP is sufficient to cause complete inactivation of the ATPase.
Bidwai
A P
AP
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill 27599.
Morjana
N A
NA
Scarborough
G A
GA
D000249:Adenosine Monophosphate / Q000378:metabolism; D001665:Binding Sites; D002462:Cell Membrane / Q000201:enzymology; D004357:Drug Synergism; D004492:Edetic Acid / Q000494:pharmacology; D008055:Lipids / Q000494:pharmacology; D008278:Magnesium Sulfate / Q000494:pharmacology; D009491:Neurospora / Q000201:enzymology*; D009492:Neurospora crassa / Q000201:enzymology*; D009711:Nucleotides / Q000378:metabolism*; D010084:Oxidation-Reduction; D010504:Periodic Acid; D006180:Proton-Translocating ATPases / Q000037:antagonists & inhibitors / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology
D008055:Lipids; D009711:Nucleotides; D010504:Periodic Acid; D014638:Vanadates; D000249:Adenosine Monophosphate; D008278:Magnesium Sulfate; D004492:Edetic Acid; D006180:Proton-Translocating ATPases