Expression, purification, and kinetic characterization of recombinant human adenylosuccinate lyase.
Adenylosuccinate adenosine 5'-monophosphate lyase (EC 4.3.2.2; ASL) catalyzes two distinct reactions in adenosine 5'-monophosphate (AMP) biosynthesis. A S413P mutation in ASL segregates with mental retardation in an affected family (Stone, R. L., Aimi, J., Barshop, B. A., Jaeken, J., Van den Berghe, G., Zalkin, H., and Dixon, J. E. (1992) Nature Genet. 1, 59-63). ASL and S413P ASL have been expressed, purified, and kinetically characterized. Lowering the Escherichia coli growth temperature to 25 degrees C and the concentration of inducer, isopropyl-1-thio-beta,D-galactopyranoside, to 40 microM was necessary for synthesis of soluble, tetrameric enzymes. The recombinant enzymes were purified to homogeneity using anion exchange chromatography followed by chromatography on Blue 2A Sepharose. At pH 7.0 and 25 degrees C, the kcat for cleavage of 5-amino-4-imidazole-N-succinocarboxamide ribotide (SAI-CAR) by ASL was 90 s-1 with a Km of 2.35 microM. The kcat for adenylosuccinate (SAMP) cleavage was 97 s-1 with a Km of 1.79 microM. The catalytic mechanism involved one general base catalyst (pK alpha = 6.4) and one general acid catalyst (pK alpha = 7.5). ASL follows an ordered uni-bi reaction mechanism with fumarate released first. 5-Amino-4-imidazolecarboxamide ribotide (AICAR) and AMP were competitive with SAICAR and SAMP (Ki[AICAR] = 11.3 microM; Ki[AMP] = 9.2 microM), whereas fumarate inhibited noncompetitively (Kii = 2.3 mM, Kis = 2.8 mM). The competitive inhibition by AICAR and AMP suggests a single active site that binds both SAICAR and SAMP. The kinetic constants at pH 7.0, 25 degrees C and the kcat/Km versus pH profiles for ASL and S413P ASL were very similar. These results are consistent with S413P being a structural rather than a catalytic defect.
Stone
R L
RL
Department of Biological Chemistry, University of Michigan Medical School, Ann Arbor 48109-0606.
Zalkin
H
H
Dixon
J E
JE
D000264:Adenylosuccinate Lyase / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism*; D000595:Amino Acid Sequence; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000378:metabolism; D001483:Base Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D015870:Gene Expression; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008607:Intellectual Disability / Q000201:enzymology / Q000235:genetics; D007700:Kinetics; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D009838:Oligodeoxyribonucleotides; D010957:Plasmids; D017354:Point Mutation; D016133:Polymerase Chain Reaction / Q000379:methods; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000378:metabolism; D013379:Substrate Specificity
D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012265:Ribonucleotides; D000620:Aminoimidazole Carboxamide; D000264:Adenylosuccinate Lyase; C020635:SAICAR