MULTINUCLEAR MAGNETIC RESONANCE STUDIES OF PHOSPHOGLUCOMUTASE (ENZYME MECHANISM, LABELING, NMR)

GYUNG IHM RHYU, Purdue University

Abstract

Rabbit muscle phosphoglucomutase has been studied by ('1)H, ('31)P, ('7)Li, and ('113)Cd NMR spectroscopy. The ('31)P NMR resonances of phosphates in intermediate species of the phosphoglucomutase reaction in the presence or absence of metal ions (glucose 1-phosphate bound to the phosphoenzyme, glucose 1,6-bisphosphate bound to the dephosphoenzyme, and glucose 6-phosphate bound to phosphoenzyme) were assigned by ('17)O-labeling of enzymic or substrate phosphate or by polarization transfer experiments. The ('31)P NMR and ('7)Li NMR studies indicated that the transfer of alternative phosphate groups from bound glucose 1,6-bisphosphate to the serine residue (116) of the dephosphoenzyme not only produces different phospho-forms of the enzyme but also different chemical environments for the metal ion bound at the activating site. NMR relaxation measurements indicated that the serine phosphate group of the phosphoenzyme is solvent accessible. Metal ion binding does not affect the solvent accessibility. The relative contributions provided by various relaxation mechanisms including ('17)O-substitution to the relaxation of the serine phosphate were deduced from the dependence of the relaxation parameters on the field strength and ('2)H(,2)O content of the solvent. The pH dependence of ('1)H NMR spectra indicated that an anion cluster is located at the metal ion binding site of the enzyme. The enzyme exists as a pH-dependent mixture of conformers that provide two different environments for the enzymic phosphate group. Cd('2+) binds directly to the enzymic phosphate group; a 16 Hz coupling constant between ('31)P and ('113)Cd nuclei in the ('113)Cd complex of the phosphoenzyme, was observed in the ('31)P NMR spectrum. In the presence of substrate the coupling disappeared. ('113)Cd NMR and ('1)H NMR studies suggested that binding of substrate causes a conformational change of the enzyme leading to a replacement of one oxygen ligand to the Cd('2+) ion (probably from the enzymic phosphate group) by one nitrogen ligand (probably from a histidine side chain of the enzyme). In addition to the binding of Li('+) and Cd('2+) at the activating site for metal ions, weaker binding of a second equivalent of either metal ion at an ancillary site was observed to occur in the presence of bound substrate.

Degree

Ph.D.

Subject Area

Biochemistry

COinS