Kinetic studies on the regulation of rabbit liver pyruvate kinase

Abstract

Two kinetically distinct forms of pyruvate kinase (EC 2.7.1.40) were isolated from rabbit liver by using differential ammonium sulphate fractionation. The L or liver form, which is allosterically activated by fructose 1,6-diphosphate, was partially purified by DEAE-cellulose chromatography to give a maximum specific activity of 20 units/mg. The L form was allosterically activated by K(+) and optimum activity was recorded with 30mm-K(+), 4mm-MgADP(-), with a MgADP(-)/ADP(2-) ratio of 50:1, but inhibition occurred with K(+) concentrations in excess of 60mm. No inhibition occurred with either ATP or GTP when excess of Mg(2+) was added to counteract chelation by these ligands. Alanine (2.5mm) caused 50% inhibition at low concentrations of phosphoenolpyruvate (0.15mm). The homotropic effector, phosphoenolpyruvate, exhibited a complex allosteric pattern (n(H)=2.5), and negative co-operative interactions were observed in the presence of low concentrations of this substrate. The degree of this co-operative interaction was pH-dependent, with the Hill coefficient increasing from 1.1 to 3.2 as the pH was raised from 6.5 to 8.0. Fructose 1,6-diphosphate interfered with the activation by univalent ions, markedly decreased the apparent K(m) for phosphoenolpyruvate from 1.2mm to 0.2mm, and transformed the phosphoenolpyruvate saturation curve into a hyperbola. Concentrations of fructose 1,6-diphosphate in excess of 0.5mm inhibited this stimulated reaction. The M or muscle-type form of the enzyme was not activated by fructose 1,6-diphosphate and gave a maximum specific activity of 0.3 unit/mg. A Michaelis-Menten response was obtained when phosphoenolpyruvate was the variable substrate (K(m)=0.125mm), and this form was inhibited by ATP, as well as alanine, even in the presence of excess of Mg(2+).

Kinetics and mechanism of action of muscle pyruvate kinase

“…The enzymes, which have been isolated from mammalian tissues (Tietz & Ochoa, 1958;Irving & Williams, 1973;Nicholas & Bachelard, 1974) (Reynard et al, 1961;Ainsworth & MacFarlane, 1973), an ordered addition of substrates and dissociation of products, for the yeast enzyme (MacFarlane & Ainsworth, 1972) and a Ping-Pong mechanism involving a phosphoenzyme for the liver L-type enzyme (MacFarlane & Ainsworth, 1974). There are difficulties, however, in the interpretation of initial-velocity studies, and we are therefore studying the mechanism of these enzymes with a variety of techniques, including flux measurements with labelled substrates (Britton, 1966;, isotope-trapping (Rose et al, 1974), binding and initial-velocity studies.…”

Pyruvate kinase from muscle and fat body of the house cricketAcheta domesticus L.: Purification and catalytic studies

“…Similar results were obtained by Wood (1968) and KorneekiGerrity and Penney (1974b) for rabbit muscle and turtle liver pyruvate kinase. The pH optimum of purified cricket PK is similar to the pH optimum for mammalian liver L-form (Irving and Williams, 1973) although lower than the pH optimum of 7.2 observed in the locust fat body and flight muscle enzyme (Bailey and Walker, 1969). As in Mytilus mantle pyruvate kinase (Livingstone and Bayne, 1974), FDP activation (in the physiological range) is independent of pH.…”

Fructose‐1,6‐bisphosphate reduces inflammatory pain‐like behaviour in mice: role of adenosine acting on A1 receptors

“…, 1991); and (iii) FBP is a step after enzymes such as hexokinase and phosphofructokinase that are regulatory points of the pathway, and before pyruvate kinase of which FBP is an allosteric activator (Taylor and Bailey, 1967;Bailey et al. , 1968; Irving and Williams, 1973). Therefore, it is possible that FBP causes an increase in the intermediates of glycolysis, which induces an efflux of AMP from the cell.…”