Expression and Biochemical Characterization of the Human Enzyme N-Terminal Asparagine Amidohydrolase

• Published in: Biochemistry (Easton) Vol. 50; no. 14; pp. 3025 - 3033
• Main Authors: Cantor, Jason R, Stone, Everett M, Georgiou, George
• Format: Journal Article
• Language: English
• Published: United States American Chemical Society 12.04.2011
• Subjects: Amidohydrolases - chemistry; Amidohydrolases - genetics; Amidohydrolases - metabolism; Animals; Asparagine - chemistry; Asparagine - metabolism; Aspartic Acid - chemistry; Aspartic Acid - metabolism; Biocatalysis - drug effects; Circular Dichroism; Cysteine - chemistry; Cysteine - genetics; Cysteine - metabolism; Electrophoresis, Polyacrylamide Gel; Humans; Hydrogen-Ion Concentration; Kinetics; Metals - pharmacology; Mice; Models, Chemical; Molecular Structure; Mutation; Recombinant Proteins - chemistry; Recombinant Proteins - metabolism; Spectrometry, Mass, Electrospray Ionization; Substrate Specificity
• ISSN: 0006-2960; 1520-4995; 1943-295X; 1520-4995
• DOI: 10.1021/bi101832w

The enzymatic deamidation of N-terminal l-Asn by N-terminal asparagine amidohydrolase (NTAN1) is a feature of the ubiquitin-dependent N-end rule pathway of protein degradation, which relates the in vivo half-life of a protein to the identity of its N-terminal residue. Herein, we report the bacterial expression, purification, and biochemical characterization of human NTAN1 (hNTAN1). We show here that hNTAN1 is highly selective for the hydrolysis of N-terminal peptidyl l-Asn but fails to deamidate free l-Asn or l-Gln, N-terminal peptidyl l-Gln, or acetylated N-terminal peptidyl l-Asn. Similar to other N-terminal deamidases, hNTAN1 is shown to possess a critical Cys residue that is absolutely required for catalysis, corroborated in part by abolishment of activity through the Cys75Ala point mutation. We also present evidence that the exposure of a conserved l-Pro at the N-terminus of hNTAN1 following removal of the initiating l-Met is important for the function of the enzyme. The results presented here should assist in the elucidation of molecular mechanisms underlying the neurological defects of NTAN1-deficient mice observed in other studies, and in the discovery of potential physiological substrates targeted by the enzyme in the modulation of protein turnover via the N-end rule pathway.