Studies on rabbit liver phosphoglucomutase

• Main Author: Jamil, Haris
• Published: Royal Holloway, University of London 1983
• Subjects: 572; Biochemistry
• Online Access: http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.704621

Phosphoglucomutases from different sources exhibit a variety of kinetic behaviour though it is probable they all have a common phosphoenzyme mechanism. The variations in behaviour arise partly from an intrinsic diphosphatase activity, which, even when present as a minor component, can affect the initial velocity patterns. Only isotopic induced-transport tests distinguish unequivocally between possible mechanisms. Rabbit liver phosphoglucomutase has a substantial amount of an isoenzyme, which appears to be associated with a high intrinsic diphosphatase activity. This activity may be involved in modulation of the glucose-1,6-diP level in the liver and hence in the regulation of carbohydrate metabolism. A procedure for the isolation of comparatively undegraded phosphoglucomutase (mol. wt. 67,600) from rabbit liver with high specific activity is described. The purified preparation showed a low diphosphatase activity. Kinetic studies, using a sensitive fluorimetric assay at ionic strength 0.05 mol 1^-1 and Mg⁺⁺ free = 2.0 mM, give a parallel line initial velocity pattern. This behaviour is consistent with the phosphoenzyme mechanism. The liver enzyme indicated a higher K_m for glucose-1,6-diP than many other phosphoglucomutases. Induced transport tests using ¹⁴C- and ³²P-labelled substrates showed that the enzyme can only have a phosphoenzyme mechanism. The isolation of ³²P-labelled phosphoenzyme which rapidly exchanged more than 95% of its ³²P-label with substrates confirmed that this phosphoenzyme is a true kinetic intermediate in the mutase reaction. Labelled phosphoenzyme contained 0.45-0.6 mol ³²P mol^-1 enzyme and its half-life was 47.5 h at 30 °C. Evidence is presented that the phosphate is bound to a serine residue of the enzyme. It is concluded that the low diphosphatase activity associated with the enzyme does not significantly modulate the level of glucose-1,6-diP in liver. Moreover, other factors must be responsible for the failure to isolate a completely phosphorylated phosphoenzyme. Although this phosphoglucomutase has a relatively high K_m for glucose-1,6-diP, the known fluctuations in the level of glucose-1,6-diP in the liver are unlikely to affect phosphogluco-mutase activity.