Pyruvate kinase was purified from cat and trout muscle. The enzymes had similar amino acid compositions and subunit molecular weights. In contrast to the mammalian enzyme, the trout muscle pyruvate kinase was activated by fructose 1,6-bisphosphate. However, unlike the 1.-type pyruvate kinase from mammalian liver it was not phosphorylated by cyclic-AMP-dependent protein kinase. The purified enzyme from cat muscle was carboxymethylated with iod0[2-'~C]acetic acid under conditions that led to the preferential labelling of one especially reactive thiol group. The labelled enzyme was cleaved with CNBr, and the radioactive fragment purified. Amino acid sequence analysis of the reactive-thiol-containing fragment from cat muscle pyruvate kinase showed it had the following sequence : Ile-Gly-Arg-['4C]CmCys-Asn-Arg-Ala-Gly-Lys-Pro-Va~-Ile-CmCys-Ala-Thr-Gln-Hse. The corresponding peptide from trout pyruvate kinase had only one difference in its amino acid composition and the sequence around the reactive thiol was identical.Pyruvate kinase is a key regulatory enzyme found in all cells and tissues during glycolysis, and catalyses the following reaction :The properties of the enzyme have been reviewed by Boyer [I] and Kayne 121. The reaction is virtually irreversible and requires a bivalent cation, such as Mg2+ or Mn2+, and a monovalent cation, normally K+. The enzyme is a tetramer with identical subunits of M , x 60000. The regulation of the activity of pyruvate kinase is important in the control of glycolysis, especially in tissues capable of gluconeogenesis. Isolation of the enzyme from different tissues has led to the identification of three major classes of isoenzymes. The L-type enzyme (first isolated from mammalian liver) shows a sigmoidal dependence of activity on P-pyruvate concentration, and is inhibited by gluconeogenic amino acids such as alanine. The affinity for Ppyruvate is enhanced by the addition of the activator, fructose 1,6-bisphosphate. The L-type enzyme is also allosterically activated by H+, and inhibited by ATP. In contrast, the M-type enzyme (first isolated from mammalian muscle) has been considered not to possess allosteric properties, although conditions have been found under which the muscle enzyme displays kinetic properties typical of a regulatory enzyme [3]. The A-type pyruvate kinase has properties intermediate between those of the M-type and L-type enzymes.M-type pyruvate kinase has been studied extensively; the X-ray crystallographic structure of the enzyme from cat muscle is known at a resolution of 0.26 nm [4]. It has been shown by low-resolution studies that each subunit has one ATPIPpyruvate/Mn2 + binding site and a secondary (non-catalytic)ADP binding site 151. Much less is known of the structure of Ltype pyruvate kinase. The liver enzyme has been shown to be phosphorylated on a specific serine residue by incubation with [32P]ATP and cyclic-AMP-dependent protein kinase, with a concomitant decrease in activity [6]. These studies show that the inhibitory effect of phosphorylation is coun...

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