Magnetic Resonance Studies on Manganese‐Activated Phosphofructokinase

Abstract

Rabbit skeletal-muscle phosphofructokinase exhibits similar kinetic behaviour and specific activity when manganese is used as the essential divalent cation in place of magnesium. At pH 7.5, the kinetics are Michaelis-Menten with respect to fructose 6-phosphate, ATP and manganous ion concentrations ; the reaction, as a function of metal ion concentration, is inhibited competitively by excess free ATP and by calcium ions. Between pH 7.0 and 6.5, in the presence of high ATP concentrations, there is a sharp transition to sigmoid kinetics as indicated by an increase of the Hill interaction coefficient from 1 to 4.1. Preincubation of the enzyme with ligands has a marked effect on activity, particularly free divalent metals which cause dissociation and an almost total loss of activity.Studies on the rates of water-proton longitudinal relaxation a t pH 7.5 and 35 MHz indicate that, in the absence of nucleotides, the phosphofructokinase-manganese system shows low relaxation enhancements (&b m 2). On titrating with ATP a t fixed enzyme and manganese ion concentrations, the enhancement rises sharply, then falls again at very high ATP levels. An analysis of enhancement data a t fixed ATP concentrations as a function of enzyme and metal ion concentrations, using a combination of graphical and computational methods, indicates that the protomer of 90000 molecular weight has 2 Mn-ATP sites with dissociation constant 240 pM and an enhancement for the ternary complex Et, of 18. Free ATP binds competitively with a dissociation constant of 3 mM. Line-broadening studies on the H-2 and H-8 resonances of the adenine ring in the high resolution spectrum of ATP reveal that the proton-manganese distances in Mn-ATP are almost unchanged in forming the ternary complex with enzyme. The kinetic and magnetic resonance data indicate that Mn-ATP is the true substrate, which probably binds to the enzyme via a nucleotide bridge.The kinetic activity of phosphofructokinase from a great variety of sources has been studied with particular regard to its role in the control of glycolysis. Studies on the enzyme from mammalian skeletal muscle have been mainly involved with its characterisation [l], subunit structure [2], the regulatory properties [3], the effects of chemical modification [4,5] and the reaction mechanism [6]. All the studies on activity have used magnesium as the essential divalent cation. The metal ion is thought to bind to the enzyme in the form of a complex with ATP which is the true substrate.A preliminary observation made in this laboratory [7] showed that the enzyme was highly active when manganese replaces magnesium as the essential diAbbreviations. NMR, nuclear magnetic resonance; ESR, electron spin resonance; Fru-6-P, fructose 6-phosphate.Enzymes. Phosphofructokinase (EC 2.7.1.11) ; creatine kinase (EC 2.7.3.2) ; pyruvate kinase(EC 2.7.2.40) ; aldolase orketose-l-phosphate aldehyde-lyase (EC 4.1.2.7) ; triosephosphate isomerase (EC 5.3.1.1); glycerol-l-phosphate dehydrogenase (EC 1.1.1.8).valent cation. This opened up the possibil...

Conformational states of rabbit muscle phosphofructokinase investigated by a spin label probe

“…In an earlier communication [4] we described the use of magnetic resonance techniques to investigate the conformation at the MnATP binding sites of phosphofructokinase; the methods suffered from complications common to the use of any dissociable. probe.…”

“…It was stored as the ammonium sulphate suspension without crystallisation and used within two months. The assays used were as described previously [4] at both 92 allosteric and non-allosteric pH, except for the omission of 2-mercaptoethanol.…”

“…From the quenching, which is distance-dependent, in conjunction with other magnetic resonance methods, a preliminary estimate of 12 A has been made for the distance between the manganous ion and the nitroxide group on the labelled enzyme. Furthermore the spin label causes an appreciable broadening of the high resolution spectra of protons in MgATP bound to the enzyme, and studies of this permit estimates of the ATP proton-nitroxide distances on the enzyme to be made which are compatible with the manganesenitroxide distance and with the previously determined structure for MnATP in the ternary complex with enzyme [4].…”

Spin‐Labelled Phosphofructokinase

“…However the main limitation of such studies is that they are based almost entirely on kinetic data. In previous work [20] we reported a simple procedure for spin-labelling phosphofructokinase specifically at its most reactive thiol group without significantly altering the allosteric kinetic behaviour or the polymerisation behaviour. Changes in the electron spin resonance (ESR) spectrum of the bound spin label on adding ligaiids were taken as an indication of conforination change in the protein, and used as preliminary evidence for the two-state mechanism of Monod et al [18].…”

“…Changes in the electron spin resonance (ESR) spectrum of the bound spin label on adding ligaiids were taken as an indication of conforination change in the protein, and used as preliminary evidence for the two-state mechanism of Monod et al [18]. This was based on the observation of two spectral components in the absence of added ligands, corresponding to mobile and immobile states of the label, the observation of spectral isosbestic points, and the effects of some activators and inhibitors on the spectra [20,21].…”