Human acetyl-coenzyme A:α-glucosaminide N-acetyltransferase. Kinetic characterization and mechanistic interpretation

Abstract

Acetyl-CoA: alpha-glucosaminide N-acetyltransferase (N-acetyltransferase) is an integral lysosomal membrane protein which catalyses the transfer of acetyl groups from acetyl-CoA on to the terminal glucosamine in heparin and heparan sulphate chains within the lysosome. In vitro, the enzyme is capable of acetylating a number of mono- and oligo-saccharides derived from heparin, provided that a non-reducing terminal glucosamine is present. We have prepared highly enriched lysosomal membrane fractions from human placenta by a combination of differential centrifugation and density-gradient centrifugation in Percoll. This preparation was used to investigate the kinetics of the enzyme with three acetyl-acceptor substrates, i.e. glucosamine and a disaccharide and a tetrasaccharide derived from heparin, each containing a terminal glucosamine residue. The enzyme showed a pH optimum at 6.5, extending to 8.0 for the mono- and di-saccharide substrates but falling off sharply above pH 6.5 for the tetrasaccharide substrate. We identified two distinct Km values for the glucosamine substrate at both pH 7.0 and pH 5.0, whereas the tetrasaccharide substrate displayed only a single Km value at each pH. The Km values were found to be highly pH-dependent, and at pH 5.0 the values for the acetyl-acceptor substrates showed a decreasing 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 analysis, isotope exchange between N-acetylglucosamine and glucosamine, and inhibition studies with desulpho-CoA indicate that the enzyme operates by a random-order ternary-complex mechanism. Product inhibition studies display a complex pattern of dead-end inhibition. Taken in context with what is known about lysosomal utilization and physiological levels of acetyl-CoA, these results suggest that in vivo the enzyme operates via a random-order ternary-complex mechanism which involves the utilization of cytosolic acetyl-CoA to transfer acetyl groups on to the terminal glucosamine residues of heparin within the lysosome.

Oral Administration of Polymer Hyaluronic Acid Alleviates Symptoms of Knee Osteoarthritis: A Double-Blind, Placebo-Controlled Study over a 12-Month Period

“…Although its mechanism is not fully understood, glucosamine is considered to improve OA symptoms and inhibit disease progression by exhibiting chondroprotective and anti-inflammatory activities [28–32]. In vivo , glucosamine is converted to N-acetyl glucosamine within cells by the actions of a series of lysosomal enzymes [33]. As mentioned earlier, N-acetyl glucosamine is the monosaccharide that comprises HA together with D-glucuronic acid.…”

Functional analysis of the HGSNAT gene in patients with mucopolysaccharidosis IIIC (Sanfilippo C Syndrome)

“…A conformational change permits the transfer of the acetyl group into the acidic environment of the lysosome. Once heparan sulphate interacts with the active site, the terminal glucosamine residue receives the acetyl group, and N-acetylglucosamine is generated [Bame and Rome, 1985, 1986a, 1986bMeikle et al, 1995]. Furthermore, the gene for this enzyme has only been recently cloned [Fan et al, 2006;Hřebíček et al, 2006].…”

Identification of the Gene Encoding the Enzyme Deficient in Mucopolysaccharidosis IIIC (Sanfilippo Disease Type C)

“…4,5 An alternative model proposes that the enzyme operates via a random-order ternary-complex mechanism; that is, there is no acetylated-enzyme intermediate. 6 The enzyme has proven very difficult to purify but is believed to be a dimer of 120 kDa subunits containing Asn-linked oligosaccharides; that is, concanavalin A-binding, with a pI of ∼7.4, based on { 14 C}acetyl-CoA labeling experiments. 4 It is also postulated that the 120-kDa subunit may only be the catalytic subunit of a protein complex whose other unidentified members are also needed for functionality.…”