Mechanism of the lysosomal membrane enzyme acetyl coenzyme A: alpha-glucosaminide N-acetyltransferase

Thesis/Dissertation · Wed Jan 01 04:00:00 EST 1986

OSTI ID:6703578

Bame, K J

Acetyl-CoA:..cap alpha..-glucosaminide N-acetyltransferase is a lysosomal membrane enzyme, deficient in the genetic disease Sanfilippo C syndrome. The enzyme catalyzes the transfer of an acetyl group from cytoplasmic acetyl-CoA to terminal ..cap alpha..-glucosamine residues of heparan sulfate within the organelle. The reaction mechanism was examined using high purified lysosomal membranes from rat liver and human fibroblasts. The N-acetyltransferase reaction is optimal above pH 5.5 and a 2-3 fold stimulation of activity is observed in the presence of 0.1% taurodeoxycholate. Double reciprocal analysis and product inhibition studies indicate that the enzyme works by a Di-Iso Ping Pong Bi Bi mechanism. The binding of acetyl-CoA to the enzyme is measured by exchange label from (/sup 3/H)CoA to acetyl-CoA, and is optimal at pH's above 7.0. The acetyl-enzyme intermediate is formed by incubating membranes with (/sup 3/H)acetyl-CoA. The acetyl group can be transferred to glucosamine, forming (/sup 3/H)N-acetylglucosamine; the transfer is optimal between pH 4 and 5. Lysosomal membranes from Sanfilippo C fibroblasts confirm that these half reactions carried out by the N-acetyltransferase. The enzyme is inactivated by N-bromosuccinimide and diethylpyrocarbonate, indicating that a histidine is involved in the reaction. These results suggest that the histidine residue is at the active site of the enzyme. The properties of the N-acetyltransferase in the membrane, the characterization of the enzyme kinetics, the chemistry of a histidine mediated acetylation and the pH difference across the lysosomal membrane all support a transmembrane acetylation mechanism.

• Research Organization: California Univ., Los Angeles (USA)
• OSTI ID: 6703578
• Country of Publication: United States
• Language: English

Related Subjects

550201* -- Biochemistry-- Tracer Techniques
59 BASIC BIOLOGICAL SCIENCES
ACETYLATION
ACYLATION
AMINES
ANIMAL CELLS
ANIMALS
BIOCHEMICAL REACTION KINETICS
BIOCHEMISTRY
BODY
CARBOHYDRATES
CELL CONSTITUENTS
CELL MEMBRANES
CHEMICAL REACTIONS
CHEMISTRY
COENZYMES
CONNECTIVE TISSUE CELLS
DIGESTIVE SYSTEM
ENZYMES
FIBROBLASTS
GLANDS
GLUCOSAMINE
HEXOSAMINES
HEXOSES
KINETICS
LABELLED COMPOUNDS
LIVER
LYSOSOMES
MAMMALS
MAN
MEMBRANES
MONOSACCHARIDES
NITROGEN TRANSFERASES
ORGANIC COMPOUNDS
ORGANOIDS
ORGANS
PRIMATES
RATS
REACTION KINETICS
RODENTS
SACCHARIDES
SOMATIC CELLS
TRANSFERASES
TRITIUM COMPOUNDS
VERTEBRATES