Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The energy-rich compound acetyl phosphate (ACP) was examined as a substrate for energy-linked reactions by the yeast plasma membrane H+-ATPase. The hydrolysis of ACP was sensitive to inhibition by vanadate with an IC50 approximately 1 microM, which is comparable to the level obtained in the presence of ATP. A Km of 8.29 +/- 0.65 mM for the hydrolysis of ACP was approximately 10-fold higher than that obtained for ATP, while Vmax values of 8.66 +/- 0.29 and 7.23 +/- 0.34 micromol Pi mg(-1) min(-1) were obtained with ATP and ACP, respectively. ACP formed a phosphorylated intermediate that was efficiently chased with hydroxylamine. Both ACP and ATP effectively protected the enzyme from trypsin-induced inactivation and formed identical tryptic digestion patterns, suggesting that ACP mimics the formation of conformational intermediates induced by ATP. However, unlike ATP, ACP was unable to drive proton transport by H+-ATPase. In addition, a pma1-S368F mutant enzyme that is highly insensitive to inhibition by vanadate in the presence of ATP was largely sensitive to vanadate in the presence of ACP. These results are interpreted in terms of a reverse, short-circuit pathway of the normal P-type ATPase kinetic pathway, in which the formation of E2P by-passes the E1P high-energy intermediate. In this pathway, ACP favors the formation of an E2P conformational state, which can interact with classical inhibitors like vanadate, but possesses insufficient free energy to drive proton transport by the H+-ATPase.
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PMID:Probing energy coupling in the yeast plasma membrane H+-ATPase with acetyl phosphate. 926 44

Membrane segment 5 (M5) is thought to play a direct role in cation transport by the sarcoplasmic reticulum Ca2+-ATPase and the Na+, K+-ATPase of animal cells. In this study, we have examined M5 of the yeast plasma membrane H+-ATPase by alanine-scanning mutagenesis. Mutant enzymes were expressed behind an inducible heat-shock promoter in yeast secretory vesicles as described previously (Nakamoto, R. K., Rao, R., and Slayman, C. W. (1991) J. Biol. Chem. 266, 7940-7949). Three substitutions (R695A, H701A, and L706A) led to misfolding of the H+-ATPase as evidenced by extreme sensitivity to trypsin; the altered proteins were arrested in biogenesis, and the mutations behaved genetically as dominant lethals. The remaining mutants reached the secretory vesicles in sufficient amounts to be characterized in detail. One of them (Y691A) had no detectable ATPase activity and appeared, based on trypsinolysis in the presence and absence of ligands, to be blocked in the E1-to-E2 step of the reaction cycle. Alanine substitution at an adjacent position (V692A) had substantial ATPase activity (54%), but was likewise affected in the E1-to-E2 step, as evidenced by shifts in its apparent affinity for ATP, H+, and orthovanadate. Among the mutants that were sufficiently active to be assayed for ATP-dependent H+ transport by acridine orange fluorescence quenching, none showed an appreciable defect in the coupling of transport to ATP hydrolysis. The only residue for which the data pointed to a possible role in cation liganding was Ser-699, where removal of the hydroxyl group (S699A and S699C) led to a modest acid shift in the pH dependence of the ATPase. This change was substantially smaller than the 13-30-fold decrease in K+ affinity seen in corresponding mutants of the Na+, K+-ATPase (Arguello, J. M., and Lingrel, J. B (1995) J. Biol. Chem. 270, 22764-22771). Taken together, the results do not give firm evidence for a transport site in M5 of the yeast H+-ATPase, but indicate a critical role for this membrane segment in protein folding and in the conformational changes that accompany the reaction cycle. It is therefore worth noting that the mutationally sensitive residues lie along one face of a putative alpha-helix.
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PMID:Structure-function relationships in membrane segment 5 of the yeast Pma1 H+-ATPase. 965 27


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