Reaction of 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine with histidine and cysteine residues in the active site of rabbit muscle pyruvate kinase.
The inactivation of rabbit muscle pyruvate kinase by 0.3 mM 5'-p-fluorosulfonylbenzoyl-1,N6-ethenoadenosine at pH 7.8 is biphasic. The first phase proceeds rapidly to yield a partially active enzyme (46% residual activity) followed by a slower rate which leads to total inactivation. The inactivation of the first phase can be reversed by addition of 20 mM dithiothreitol, whereas the second phase is unaffected. These two phases have second-order rate constants of 250 M-1 X min-1 (dithiothreitol-sensitive reaction) and 52 M-1 X min-1 (dithiothreitol-insensitive reaction), respectively. Marked protection against inactivation is afforded by phosphoenolpyruvate and by metal-nucleotide complexes in the presence of free metal, indicating that reaction occurs in the region of the active site. Loss of approximately two sulfhydryls per enzyme subunit correlates well with the dithiothreitol-sensitive inactivation, suggesting that this phase of the inactivation may be attributable to disulfide formation. Incorporation of about one mole of fluorescent reagent per enzyme subunit correlates closely with the dithiothreitol-insensitive phase of inactivation, yielding a modified histidine residue. The quantum yield of the fluorescent sulfonylbenzoyl-1,N6-ethenoadenosine-pyruvate kinase is only 0.007, as compared to 0.54 for the parent nucleoside 1,N6-ethenoadenosine. The quenched fluorescence is consistent with stacking of the sulfonylbenzoyl moiety on the purine ring in the modified enzyme, which suggests that the altered histidine may be located in the adenine region of the metal-nucleotide binding site.
D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D003545:Cysteine / Q000378:metabolism*; D004229:Dithiothreitol / Q000494:pharmacology; D005456:Fluorescent Dyes / Q000378:metabolism; D006639:Histidine / Q000378:metabolism*; D007700:Kinetics; D008433:Mathematics; D009132:Muscles / Q000201:enzymology*; D011770:Pyruvate Kinase / Q000378:metabolism*; D011817:Rabbits; D013777:Tetrazoles / Q000494:pharmacology
D005456:Fluorescent Dyes; D013777:Tetrazoles; C007314:5-diazo-1H-tetrazole; D006639:Histidine; C029342:5'-(4-fluorosulfonylbenzoyl)-1,N(6)-ethenoadenosine; D011770:Pyruvate Kinase; D000241:Adenosine; D003545:Cysteine; D004229:Dithiothreitol