Stereochemical aspects of the interaction between isomerases and their substrates

The work presented in this thesis is concerned with the mechanism of two enzymes of the glycolytic pathway, each of which catalyzes an aldose-ketose isomerization.
The first part of the thesis deals with triosephosphate isomerase, which catalyzes the interconversion of dihydroxyacetone phosphate and D-glyceraldehyde 3-phosphate. A mechanism has been proposed for this reaction that involves the transfer of a proton between carbon centres of the substrates, and an enzyme-bound cis-enediol as a reaction intermediate.1
The orientation and accessibility of the enzyme-bound substrates was studied by boro[3H]hydride reduction of unlabelled triosephosphates and borohydride reduction of monodeuterated substrates both in the presence and absence of a large concentration of the isomerase. Analysis of the distribution of the hydrogen isotope in the reduction product, glycerol phosphate showed that of the four possible positions of borohydride attack on the enzyme-bound species (from the si and re face of each of the substrate carbonyl groups), only one product is formed: that resulting from reduction at the si face of dihydroxyacetone phosphate. Indeed, reduction occurs here some 14-times faster than at the si face of this substrate in free solution. The inaccessibility of the other three faces of the carbonyl groups of the enzyme-bound substrates is presumably due to the steric bulk of the enzyme. Reduction from the si face of dihydroxyacetone phosphate is actually catalyzed by the isomerase in spite of any expected inaccessibility. This catalysis is best explained by the existence of an electrophilic group at the enzyme active site that polarizes the carbonyl group of dihydroxyacetone phosphate. It is proposed that this electrophilic group is in part responsible for the facile abstraction of the proton from the substrate in the enzyme-mediated isomerization.
In order to obtain direct evidence for the cis-enediol intermediate of the isomerase-catalyzed reaction, deuterated diimide was used in an attempt to trap this species. Since diimide reduces only non-polar double bonds, it was expected to reduce the enediol in a syn manner to give doubly-labelled glycerol phosphate. The stereochemistry of the enediol could then be determined from a knowledge of the position of labelling in the glycerol phosphate product. Unfortunately the yield of glycerol phosphate was too small to perform the necessary stereochemical analysis.
Each of the triose phosphates exists in two forms: the hydrate and keto form.2,3 The keto forms are known to be the true substrates of the isomerase,2,3 but the kinetic and equilibrium parameters of the catalyzed reaction must be modified because a proportion of the substrate is present as the inactive hydrate. These parameters would have to be further modified if the hydrates were to bind to the enzyme as competitive inhibitors. This question was investigated by n.m.r. spectroscopy.
Using proton n.m.r., the percentage of dihydroxyacetone phosphate present as the hydrate was found to increase on decreasing the temperature from 23°C to 1°C, or on protonating the substrate. Binding of hydrate and keto forms of dihydroxyacetone. phosphate to the isomerase was studied by measurement of peak widths in the phosphorus n.m.r. spectra at increasing enzyme concentrations. At pH 6.9 and 1°C, the keto peak was considerably broadened due to binding of this species, whereas no broadening of the hydrate peak was observable even at high enzyme concentrations. This indicates that the hydrate binds to the isomerase at least one order magnitude less tightly than does the keto form.
The second part of this thesis, is concerned with glucose-6-phosphate isomerase, which catalyzes the interconversion of glucose 6-phosphate and fructose 6-phosphate. The mechanism proposed for this interconversion is analogous to that for triosephosphate isomerase, namely the transfer of a proton between carbon centres of the keto forms of the substrates via a cis-enediol reaction intermediate.4,5 The α-anomer of each substrate is used preferentially, with the β-anomer being isomerized at a somewhat slower rate.6,7 The isomerase also catalyzes the anomerization of each substrate,6,7 but it is not clear how the two activities of the enzyme are related mechanistically. In principle, the use of deoxy analogues of the substrates should allow each enzyme activity to be studied independently from the other, and in this thesis experiments are described that test the feasibility of this approach.
2-Deoxyglucose 6-phosphate and 1-deoxyfructose 6-phosphate are close analogues of the cyclic forms of the substrates. They cannot undergo aldose-ketose isomerization but may undergo enzyme-catalyzed anomerization. A preparation of 1-deoxyfructose 6-phosphate was developed and its Ki-value with the isomerase was determined to be 0.3 mM at 30° and pH 7.5.
The isomerase catalyzes the incorporation of tritium into 2-deoxyglucose 6-phosphate from tritiated water at a rate some 106-times slower than into the natural substrate. This enzyme-catalyzed tritium exchange may be explained by enolization of the analogue on the surface of the enzyme in a manner similar to the formation of the enediol in the normal enzymic reaction. No tritium exchange could be observed to occur into 1-deoxyfructose 6-phosphate.
Since these analogues bind to the isomerase with values of Ki similar to the Michaelis constants of the natural substrate, but undergo enolization extremely slowly on the enzyme, they promise well for studies of the anomerization reaction of glucose-6-phosphate isomerase.
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