Adenylosuccinate synthetase from Escherichia coli is inactivated in a biphasic reaction by guanosine 5'-O-[S-(4-bromo-2,3-dioxobutyl)thio]phosphate (GMPSBDB) at pH 7.1 and 25 degrees C. Reaction of the enzyme with [8-3H]GMPSBDB results in the incorporation of 2 mol of the reagent/mol of subunit; in the presence of active site ligands the incorporation is reduced to 1 mol of reagent/mol of subunit. GMPSBDB reacts with Cys-291 in the initial rapid reaction which is accompanied by loss of 50% of the enzymatic activity; this reaction is not affected by the presence of active site ligands. In the slower reaction, GMPSBDB inactivates the enzyme by reacting with Arg-143. The inactivation kinetics of the slower phase are consistent with the formation of an enzyme--GMPSBDB complex having a Kd of 42 microM. Active site nucleotides, either adenylosuccinate or IMP + GTP, prevent both slower phase inactivation and labeling of Arg-143. Replacement of Arg-143 with a Leu by site-directed mutagenesis does not change the catalytic constant or the K(m) for aspartate but does significantly impair nucleotide binding: the Michaelis constants for IMP and GTP increase by 60-fold and 10-fold, respectively, in the R143L mutant. The crystal structure of the E. coli enzyme [Poland, B.W., Silva, M.M., Serra, M.A., Cho, Y., Kim, K. H., Harris, E.M.S., & Honzatko, R.B. (1993) J. Biol. Chem. 268, 25334--25342] shows that Arg-143 from one subunit projects into the putative active site of the other subunit. These results indicate that both subunits of dimeric adenylosuccinate synthetase contribute to each active site and that Arg-143 plays an important role in nucleotide binding.

NAD(P)H/quinone acceptor oxidoreductase type 1 (QR1) protects cells from cytotoxic and neoplastic effects of quinones though two-electron reduction. Kinetic experiments, docking, and binding affinity calculations were performed on a series of structurally varied quinone substrates. A good correlation between calculated and measured binding affinities from kinetic determinations was obtained. The experimental and theoretical studies independently support a model in which quinones (with one to three fused aromatic rings) bind in the QR1 active site utilizing a π-stacking interaction with the isoalloxazine ring of the FAD cofactor. NAD(P)H:quinone acceptor oxidoreductase type 1 (QR1, 1 EC 1.6.99.2), a homodimeric flavoprotein of 273 residues (M ) 30815) per monomer, catalyzes an obligatory twoelectron reduction of quinones using either NADH or NADPH as electron donors (1, 2). QR1 affords protection against the cytotoxic and neoplastic effects of electrophilic quinones and partially reduced semiquinones that can undergo redox cycling to generate active oxygen species (3). QR1, found in many solid tumors at elevated levels, can be used to target the tumor cells through bioreductive activation of quinone-based chemotherapeutic agents (3).The crystal structures of quinone reductases from rat (PDB ID: 1QRD), mouse (1DXQ), and human (1DXO, 1D4A), reflecting a high degree of sequence identity, show very small differences in the positions of the 220 R-carbons in the catalytic domain and possess nearly identical active sites (1,4,5). The crystal structure for the rat enzyme (1QRD) contains duroquinone, FAD, and the NAD(P)H analogue cibracon blue, but the latter ligand sterically intrudes into the active site, affecting the position of at least one key residue, Tyr128 (1, 4). The human enzyme, whose crystal structure (1DXO) contains duroquinone and FAD but no cibracon blue, differs from the rat enzyme by one amino acid (Gln104 in human, Tyr104 in rat) in the active site, resulting in a slight change (0.7 Å) in the positioning of the flavin ring (1).The quinone reductase active site, produced by residues from each subunit of the dimeric protein, has the following features: (1) the duroquinone overlays the FAD isoalloxazine ring that is bound to one subunit, with the quinoid carbonyl oxygens oriented roughly parallel to the length of the flavin ring; (2) a loop from the second subunit, which forms a lid for the active site above the bound quinone, provides two tyrosine residues, Tyr126 and Tyr128, that hydrogen bond directly, or indirectly via a water molecule, to the quinone carbonyl groups; and (3) three aromatic residues provide hydrophobic contacts with the quinone, and one of these, Trp105, forms a wall bordering one side of the active site (1). A depiction of the human quinone reductase (1DXO) active site is shown in Figure 1.Mechanistically, a hydride ion from NAD(P)H is thought to be transferred to the flavin nitrogen, N 5 , with possible charge stabilization from a catalytic triad involving the FAD, Tyr155, ...

Potentials for the one-electron reduction of a series of substituted benzo-and naphthoquinones were measured in 10 aprotic solvents by cyclic voltammetry and used to consmct Hammett plots. Hammett reaction constants, p, were determined in each solvent and used as indicators of the solvent-mediated stabilization of the charged radical-anion product of the reduction reaction. Correlations of Hammett p values with solvent parameters suggest that the Lewis acidity of the solvent, in particular the solvent's ability to donate a partially positive proton, is a consistent predictor of the degree of charge stabilization of the quinone radical anion. Other mechanisms of charge stabilization, including solvent-solute charge transfer complexes and dipole-dipole interactions involving the molecular dipole moment (or an induced dipole moment) of the solvent, were found to be inconsistent predictors of the effect of solvent on one-electron quinone reduction in aprotic media.