HUMAN ACETYL-COENZYME A-ALPHA-GLUCOSAMINIDE N-ACETYLTRANSFERASE - KINETIC CHARACTERIZATION AND MECHANISTIC INTERPRETATION

Acetyl-CoA:alpha-glucosaminide N-acetyltransferase (N-acetyltransferas e) is an integral lysosomal membrane protein which catalyses the trans fer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, th e enzyme is capable of acetylating a number of mono- and oligo-sacchar ides derived from heparin, provided that a non-reducing terminal gluco samine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centri fugation and density-gradient centrifugation in Percoll. This preparat ion was used to investigate the kinetics of the enzyme with three acet yl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetr asaccharide derived from heparin, each containing a terminal glucosami ne residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 fo r the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct K-m values for the glucosamine substrate at both pH 7.0 and pH 5.0, wh ereas the tetrasaccharide substrate displayed only a single K-m value at each pH. The K-m values were found to be highly pH-dependent, and a t pH 5.0 the values for the acetyl-acceptor substrates showed a decrea sing trend as the size of the substrate increased, suggesting that the enzyme recognizes an extended region of the non-reducing terminus of the heparin or heparan sulphate polysaccharides. Double-reciprocal ana lysis, isotope exchange between N-acetylglucosamine and glucosamine, a nd inhibition studies with desulpho-CoA indicate that the enzyme opera tes by a random-order ternary-complex mechanism. Product inhibition st udies display a complex pattern of dead-end inhibition. Taken in conte xt with what is known about lysosomal utilization and physiological le vels of acetyl-CoA, these results suggest that in vivo the enzyme oper ates via a random-order ternary-complex mechanism which involves the u tilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.