Mechanistic studies on myo-inositol monophosphatase from bovine brain
Mechanistic studies on myo-inositol monophosphatase from bovine brain
Two enzyme mechanisms have been proposed for myo-inositol mono phosphatase, a ping pong bi bi mechanism and compulsory ordered mechanism. In order to determine by which mechanism the enzyme operated, the enzyme was purified to homogeneity from bovine brain. The purification was achieved in six steps, with an overall yield of 3% activity. The recombinant bovine brain myo-inositol monophosphatase enzyme was purified from Escherichia coli in five steps, with an overall yield of 26% activity. Using a range of criteria, the recombinant enzyme was shown to be identical to the myo-inositol monophosphatase isolated directly from bovine brain.
Using phosphate as the substrate, it was demonstrated that 18O-label exchange from [18O]-H2O into the phosphate pool only occurred in the presence of myo-inositol. As for the physiological reaction, Mg2+ was an essential cofactor. Extrapolation of the double reciprocal plot of the initial velocity of exchange and the concentration of myo-inositol, gave a KM value for myo-inositol of ~200 mM. Vm^x for the exchange was ~60% of Vmax for the hydrolysis reaction. Lithium cation was shown to inhibit the I8O-label exchange reaction, with an estimated K( value of ~1 mM. Using these results a new model for the myo-inositol monophosphatase enzyme mechanism was constructed.
The substrate analogue, myo-inositol 1-phosphorothioate, and its (+)-and (-)-enantiomers, were synthesised in good yield from 2,3,4,5, 6-pentakis-O-benzyl myo-inositol. It was shown that each of the compounds was a substrate for myo-inositol monophosphatase using 1H-n.m.r spectroscopy to follow the hydrolysis reaction. The substrate analogues were also shown to be competitive inhibitors for myo-inositol 1-phosphate, with Kj values of 1.1, 0.8 and 1.3 mM for the racemic and chiral forms respectively. An alternative synthesis of L-myo-inositol 1-phosphorothioate, which incorporated the enzymic reaction of myo-inositol monophosphate synthase with the substrate analogue D-glucose 6-phosphorothioate, was also investigated. D-glucose 6-phosphorothioate which was synthesised was not a substrate for the synthase. This rather curious result, which remains un-rationalised, prevented the synthesis of L-myo-inositol 1-phosphorothioate, chiral at phosphorus. Chiral phosphorothioate was to be used for the analysis of the stereochemical course of the myo-inositol monophosphatase reaction.
myo-inositol 1-phosphorothioate was also synthesised 88S-labeIled, and was used in experiments designed to isolate the putative phosphoenzyme intermediate. Initial results from these experiments were promising, although the phosphoenzyme was not characterised prior to the conclusion of this study.
University of Southampton
1991
Baker, Graham Rowland
(1991)
Mechanistic studies on myo-inositol monophosphatase from bovine brain.
University of Southampton, Doctoral Thesis.
Record type:
Thesis
(Doctoral)
Abstract
Two enzyme mechanisms have been proposed for myo-inositol mono phosphatase, a ping pong bi bi mechanism and compulsory ordered mechanism. In order to determine by which mechanism the enzyme operated, the enzyme was purified to homogeneity from bovine brain. The purification was achieved in six steps, with an overall yield of 3% activity. The recombinant bovine brain myo-inositol monophosphatase enzyme was purified from Escherichia coli in five steps, with an overall yield of 26% activity. Using a range of criteria, the recombinant enzyme was shown to be identical to the myo-inositol monophosphatase isolated directly from bovine brain.
Using phosphate as the substrate, it was demonstrated that 18O-label exchange from [18O]-H2O into the phosphate pool only occurred in the presence of myo-inositol. As for the physiological reaction, Mg2+ was an essential cofactor. Extrapolation of the double reciprocal plot of the initial velocity of exchange and the concentration of myo-inositol, gave a KM value for myo-inositol of ~200 mM. Vm^x for the exchange was ~60% of Vmax for the hydrolysis reaction. Lithium cation was shown to inhibit the I8O-label exchange reaction, with an estimated K( value of ~1 mM. Using these results a new model for the myo-inositol monophosphatase enzyme mechanism was constructed.
The substrate analogue, myo-inositol 1-phosphorothioate, and its (+)-and (-)-enantiomers, were synthesised in good yield from 2,3,4,5, 6-pentakis-O-benzyl myo-inositol. It was shown that each of the compounds was a substrate for myo-inositol monophosphatase using 1H-n.m.r spectroscopy to follow the hydrolysis reaction. The substrate analogues were also shown to be competitive inhibitors for myo-inositol 1-phosphate, with Kj values of 1.1, 0.8 and 1.3 mM for the racemic and chiral forms respectively. An alternative synthesis of L-myo-inositol 1-phosphorothioate, which incorporated the enzymic reaction of myo-inositol monophosphate synthase with the substrate analogue D-glucose 6-phosphorothioate, was also investigated. D-glucose 6-phosphorothioate which was synthesised was not a substrate for the synthase. This rather curious result, which remains un-rationalised, prevented the synthesis of L-myo-inositol 1-phosphorothioate, chiral at phosphorus. Chiral phosphorothioate was to be used for the analysis of the stereochemical course of the myo-inositol monophosphatase reaction.
myo-inositol 1-phosphorothioate was also synthesised 88S-labeIled, and was used in experiments designed to isolate the putative phosphoenzyme intermediate. Initial results from these experiments were promising, although the phosphoenzyme was not characterised prior to the conclusion of this study.
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Published date: 1991
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Local EPrints ID: 460368
URI: http://eprints.soton.ac.uk/id/eprint/460368
PURE UUID: 996ba861-5946-48c2-968a-15efe7f0b5c2
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Date deposited: 04 Jul 2022 18:20
Last modified: 04 Jul 2022 18:20
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Author:
Graham Rowland Baker
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