BBMB Research Seminars
April 19
Danny Binkowski
Department of Biochemistry, Biophysics & Molecular Biology
Iowa State University
“Kinetic studies of bacterial and human SAICAR synthetase”
Abstract:
In steps 6 and 7 of mammalian de novo nucleotide biosynthesis, a bifunctional enzyme converts 5-aminoimidazole ribonucleotide (AIR) to 4-carboxyl-5-aminoimidazole ribonucleotide (CAIR), and then CAIR to 5-aminoimidazole-4(N-succinylcarboxamide) ribonucleotide (SAICAR). This and other enzymes of purine nucleotide biosynthesis are targets in the growth inhibition of T-cell Acute Lymphoblastic Lymphomas and of microorganisms injurious to human health. Initial velocity and inhibition kinetics are data consistent with a sequential steady state Ordered kinetic mechanism for human SAICAR synthetase activity, with CAIR binding first, ATP second and l-aspartate last. The kinetic mechanism for the human enzyme differs markedly from the rapid equilibrium Random mechanism reported for Escherichia coli SAICAR synthetase, and is consistent with a CAIR-induced conformational change that orders the active site of the human enzyme. Furthermore, it is also consistent with the chemical mechanism as determined by positional isotope exchange (PIX). Suppression of interfering AIR carboxylase/CAIR decarboxylase activity in the human enzyme employed either a directed mutation (Lys304→Ala) of the AIR carboxylase site or specific ligation of the AIR carboxylase pocket by a slow, tight-binding inhibitor (called NAIR). The Michaelis constant for CAIR differs tenfold depending on whether the elimination of AIR carboxylase activity is by inhibition or by mutation. Ligation of the AIR carboxylase site by NAIR slows the rate of association of CAIR with the SAICAR synthetase pocket, indicating a functional linkage between the two active sites that maintains constant flux through SAICAR synthetase.
Publications:
Binkowski, D.J., Shen, L., Ginder, N.D., Fromm, H.J., Honzatko, R.B. Recognition of L-Aspartate and L-Alanosine by Phosphoribosyl-Aminoimidazole-Succinocarboxamide Synthetase Involves the 5'-Phosphoryl Binding Site for 5-Amino-4carboxyimidazole Ribonucleotide. (pending submission) (2007).
Binkowski, D.J., Ginder, N.D., Fromm, H.J., Honzatko, R.B. Kinetic mechanism of Human SAICAR Synthetase. (pending submission) (2007).
Binkowski, D.J., Chen, X., Ginder, N.D., Fromm, H.J., Honzatko, R.B. Chemical Mechanism of Escherichia coli and Human SAICAR Synthetase. (in preparation) (2007).
Ginder, N.D., Binkowski, D.J., Fromm, H.J. & Honzatko, R.B. Nucleotide complexes of Escherichia coli phosphoribosylaminoimidazole succinocarboxamide synthetase. J Biol Chem 281, 20680-8 (2006).
Nelson, S.W., Binkowski, D.J., Honzatko, R.B. & Fromm, H.J. Mechanism of action of Escherichia coli phosphoribosylaminoimidazolesuccinocarboxamide synthetase. Biochemistry 44, 766-74 (2005).
