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 Vmax that is only 0.012% that for glucose. While Vmax 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 Ki 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 31P 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 31P NMR chemical shifts of these analogues are 8-8.5 ppm at pH 9.5. Their relative stability is 6-aminogluconate-6-P greater than 3-aminoglyceraldehyde-3-P greater than 6-aminoglucose-6-P greater than 6-aminofructose-1,6-bis-P congruent to 6-aminofructose-6-P greater than 5-aminoribulose-5-P. These analogues were tested as substrates for their respective enzymes.(ABSTRACT TRUNCATED AT 250 WORDS)