Glucose-6-phosphatase substrate specificity

It was Fujita and his colleagues (1969) who established the unique substrate specificity of -dependent p-nitrophenylphosphata e. For example, the enzyme did not hydrolyze -glycerophosphate or phenylphos-phate, which are the standard substrates for acid and alkaline phosphatases, nor did it hydrolyze ATP, AMP, 2 -(3 )-AMP, CMP, IMP, GMP, glucose-6-phosphate, or glucose-l-phosphate. From the noncompetitive nature of inhibition of both enzyme activities in the presence of ATP and p-nitrophenylphosphate, Fujita suggests diat there are two different active sites which may or may not be located on the same protein molecule. 

By analysis of the products with glucose oxidase, it was shown that the anomeric composition of the glucose liberated from glucose-6-phos-phate by the enzymes acid or alkaline phosphatase or by glucose-6-phosphatase from rat liver was essentially the same as that of the substrate, thus indicating a lack of anomeric specificity for these enzymes also (106). 

The action of phosphatase on glucose-l-phosphate substrate is shown in Fig. 18. Phosphatase breaks the P-0 bond and releases the P04 group. In contrast phosphorylase which operates on the same substrate opens the C—0 bond. Apparently this affinity to specific bond sites, which in the present case amounts to a difference of ca. 1.5 A (Fig. 18) between the two bonds left and right from the oxygen, is... 

The third enzyme in the pathway, KD0-8-phosphate phosphatase, has been purified to homogeneity (26). Because of its abosolute specificity, it should be a focal point for chemotherapeutic studies. jThe apparent for KD0-8-phosp te was+ etermined to be 5.8 x 10 M in the presence of 1.0 mM Co or Mg. This specific KD0-8-phosphate phosphatase was separated from enzymes, present in crude extracts, having phosphatase activity on other phosphorylated compounds by column chromatography on DGAE-Sephadex (26). Three distinct peaks of activity were detected. Fractions from each peak were pooled and the rates for the hydrolysis of five compounds were measured. Peak A possessed phosphatase activity for D-glucose-6-phosphate, D-arabinose-5-phosphate, D-ribose-5-phosphate and j-nitrophenylphosphate Peak B dephosphorylated D-arabinose-5-phosphate, D-ribose-5-phosphate and D-glucose-6-phos-phate. Peak C, which was well separated from the other two peaks, could only utilize KD0-8-phosphate as a substrate. KD0-8-phos-phate was not hydrolyzed by the phosphatases present in peaks A and B. 

Eisenberg, F.J., 1967, d-myo inositol 1-phosphate as product of cyclization of glucose 6-phosphate and substrate for a specific phosphatase in rat testis. J. Biol. Chem. 242 1375-1382. 

Phosphatases specific for such substrates as glucose-6-phosphate, fructose-l,6-bisphosphate, and phospho-glycolate help to drive metabolic cycles (Chapter 17). The 335-residue fructose-1,6-bisphosphatase associates to form a tetramer with D2 symmetry. ° The allosteric enzyme exists in two conformational states (see Chapter 11). Activity is dependent upon Mg + or other suitable divalent cation, e.g., Mn or Zn, and is further enhanced by K+ or NH3. While the dimetal sites depicted in Figs. 12-23 and 12-24 are quite rigid and undergo little change upon formation of complexes with substrates or products, the active site of fructose-1,6-bisphosphatase is more flexible. There are three metal-binding sites but they contain no histidine side chains and have been seen clearly only in a product complex. Perhaps because of the need for... 

We have developed preparative enzymatic syntheses of several unusual hexoketoses using fructose-1,6-diphosphate aldolase (FDP-aldolase, E.C.4.1.2.13) as catalyst and dihydroxyacetone phosphate (DHAP) and an aldehyde as substrates (15). The enzyme appears to be very specific for DHAP but will accept a variety of aldehydes as acceptors. The ketose-1-phosphates prepared are converted to the phosphate free ketoses after removal of the phosphate group by acid- or phosphatase-catalyzed hydrolysis. The ketoses can be isomerized stereospecifically to aldoses catalyzed by glucose isomerase (E.C.5.3.1.5.) from Flavobacteriuum arborescens. The equilibrium mixtures of aldoses and ketoses are then separated by chromatography on Dowex 50 (Ba ) or Dowex 1 (HSO "). Figure 1 illustrates the preparation of a mixture of 6-deoxy-6-fluoro-D-fructose... 

However, the acid phosphatase activity of rat iiver lysosomes has recently been resolved into at least two enzymes [531]. Acid phosphatase is used in subcellular fractionation studies as a marker enzyme for lysosomes. Both acid phosphatase [E.C. 3.1.3.2] and alkaline phosphatase [E.C. 3.1.3.1] activities should not be confused with other specific phosphatases with high specificity requirements for substrate, e.g. glucose-6-phos-phatase, fructose-l,6-diphosphatase, phosphatidate phosphatase. Several assay procedures are available, u.v. estimation can be achieved using phosphoenolpyruvate as a substrate and lactate dehydrogenase in an indicator reaction [539]. Colorimetric assays can be based upon the liberation of phenol from phenylphosphate [540], upon the Uberation of phosphate from sodium /3-glycerophosphate [541], upon the hydrolysis of sodium phenolphthalein phosphate [542], or upon the hydrolysis of p-nitrophenyl phosphate [543]. 

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