PHOSPHORYLATED AMINOSUGARS - SYNTHESIS, PROPERTIES, AND REACTIVITY IN ENZYMATIC-REACTIONS

A number of phosphorylated aminosugars have been prepared and tested as substrates for metabolic reactions. 6-Aminoglucose is a slow substrate for yeast hexokinase with a V(max) that is only 0.012% that for glucose. While V(max) is pH independent, V/K decreases below the pK of 9.0 of the amino group. 6-Aminoglucose is a competitive inhibitor vs glucose with a K(i) value increasing below the pK of 9 but leveling off at 33 mM below pH 7.16. Thus, protonation decreases binding affinity by 2.4 kcal/mol and only the neutral amine is catalytically competent. 6-Aminoglucose-6-P was synthesized enzymatically with hexokinase. Its pK's determined by P-31 NMR were 2.46 and 8.02 (alpha-anomer) and 2.34 and 7.85 (beta-anomer), with a beta:alpha-ratio of 3.0. It is most stable at pH 12 (half-life 228 h at 22-degrees-C), while as a monoanion its half-life is 3 h. The free energy of hydrolysis at 25-degrees-C and pH 9.25 is -10.3 kcal/mol. The phosphorylated amino analogues of 6-P-gluconate, ribulose-5-P, fructose-6-P, fructose-1,6-bis-P (amino group at C-6 only), and glyceraldehyde-3-P were synthesized enzymatically. The P-31 NMR chemical shifts of these analogues are 8-8.5 ppm at pH 9.5. Their relative stability is 6-aminogluconate-6-P > 3-aminoglyceraldehyde-3-P > 6-aminoglucose-6-P > 6-aminofructose-1,6-bis-P approximately equals 6-aminofructose-6-P > 5-aminoribulose-5-P. These analogues were tested as substrates for their respective enzymes. Phosphoglucomutase and alkaline phosphatase were tolerant of the O to N substitution, acting on 6-aminoglucose-6-P with V(max) values 9 and 92% those for glucose-1-P and glucose-6-P. Of enzymes that do not transfer the phosphoryl group, phosphoglucoisomerse is least tolerant of the O to N change (V(max) 17% that for glucose-6-P). Phosphorylated amino analogues were good substrates for phosphofructokinase, aldolase, and glucose-6-P and 6-P-gluconate dehydrogenases. Phosphorylated aminosugars are thus excellent isosteric analogues of normal metabolic intermediates, except for reactions catalyzed by kinases.