Abstract
Reaction of the adenine nucleotide analogue 5'-p-fluorosulfonylbenzoyl adenosine at distinct tyrosine and cysteine residues of rabbit muscle pyruvate kinase.
Highlights
2.1 mM 5’-p-fluorosulfonylbenzoyladenosine (5”FSBA) been located with any certainty and, therefroerlea,tively little at pH 7.4
ADP plus M&+ provides almost complete protec- pyruvate kinase. They did not identify the amino tion against the dithiothreitol-insensitiveinactivation, acid residues attackedby 5“p-fluorosulfonylbenzoyladenosine suggesting that thetyrosine is located within the metal- nor did they distinguish the effects of substrates on reaction nucleotide binding site
Free M&’ alone prevents totally the slow dithiothreitol-sensitive inactivation indicating that the essential cysteine residues are located at or near the free mined from its absorbance a t 280 nm using E>,”,= 5.4 [24] and a molecular weight of 237,000 for the tetramericenzyme [25].Tritiated 5”p-fluorosulfonylbenzoyl adenosine (5”[’H]FSBA) was prepared from [2-’HH]adenosine (New England Nuclear Co.) by the procedure of Wyatt and Colman [23]
Summary
L times and added to 0.7-ml solution of200 mM Tris/chloride buffer, pH 8.0, containing 1.4%sodium dodecyl sulfate. 1. Inactivation of pyruvate kinase by 5'-FSBA and efreagent was separated as described for the measurement of radioac- fect of dithiothreitol on modified enzyme. Of until 55 rnin, when it was changed to 0.2 M sodium citrate buffer, pH 2 pl were withdrawn a t each time and assayed for pyruvate kinase. 4.12; the final buffer, 0.2 M sodium citrate, pH 6.4, was used to elute activity by measuring the decrease of phosphoenolpyruvate at 230 the column from 76 to 165 min Under these conditions, 0-(4-carbox- nm, as described under "Experimental procedures." Line 2, residual ybenzenesulfony1)-tyrosineappeared at 104.5 min. Calculation of the pseudo-frstorder rate constant for lysine eluted at 89 min with the use of the buffer change schedule the fast phaseof line 1 (total inactivation), was based on the equation described by Likos et al [20].
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