Daniel C. Vellom

Luminescent immobilized enzyme test systems for inorganic pyrophosphate: Assays using firefly luciferase and nicotinamide-mononucleotide adenylyl transferase or adenosine-5′-triphosphate sulfurylase

Inorganic pyrophosphate was measured by luminescence produced by a pyrophosphatase (NAD adenylyl-transferase or ATP sulfurylase) coimmobilized with firefly luciferase on Sepharose beads, with continuous flow of saturating concentrations of substrates (NAD plus luciferin or adenylophosphosulfate plus luciferin, respectively) and intermittent injections of samples containing pyrophosphate. In this scheme, the limiting substrate (pyrophosphate) is regenerated, a situation that is well suited to a bioluminescent assay. The instrumentation allowed for automation with a through-put of approximately one sample every 4 min. With standard solutions or samples that do not contain ATP, the sensitivity of the assay permits detection of less than 1 pmol pyrophosphate in a volume of 20 microliters (50 nmol/liter) with a coefficient of variation approximately equal to 4%. To assay biological samples, it was shown that endogenous ATP can be inactivated by oxidation with sodium periodate. Periodate treatment and quenching engenders dilution that limits the sensitivity to approximately 600 nmol/liter pyrophosphate in the starting material. The assay has been applied to the determination of intracellular pyrophosphate in human lymphocytes and to the measurement of nucleoside-triphosphate pyrophosphohydrolase in human fibroblasts. The variability of the assay was greater with biological samples than with standard samples, with a coefficient of variation of 15.3% in a series of determinations of intracellular pyrophosphate in a series of replicate lymphocyte lysates. Bioluminescent systems of coupled coimmobilized enzymes offer great promise for sensitive, safe, automated assaying of metabolites.

The Function of Electrostatics in Acetylcholinesterase Catalysis

A hallmark of acetylcholinesterase (AChE) catalysis is its great speed (Quinn, 1987; Rosenberry, 1975). For example, Nolte et al. (1980) reported that kcat/Km for Electrophorus electricus AChE (EeAChE) catalyzed hydrolysis of acetylthiocholine (ATCh), extrapolated to zero ionic strength, approaches 1010 M−1 s−1, a value well above the Eigen limit for bimolecular combination of ligands with enzymes (Eigen & Hammes, 1963). In addition, bimolecular rate constants that approach 1011 M−1 s−1 for binding of aromatic quaternary ammonium inhibitors have been observed (Nolte et al., 1980). The molecular origins of these high rate constants have only recently become apparent. Ripoll et al. (1993) utilized the X-ray structure of Torpedo californica AChE (TcAChE; Sussman et al., 1991) to calculate the electric field of the enzyme. They found that the hemisphere of AChE that contains the active site gorge (Sussman et al., 1991) has a high negative electrical potential, and that the overall protein has a dipole moment of greater than 500 D, with the dipole moment vector aligned with the axis of the gorge. This electric field should accelerate the binding of positive charged quaternary ammonium ligands, an idea that is supported by both theoretical calculations (Tan et al., 1992) and measurements of ionic strength effects (Nolte et al., 1980).

Amino Acid Residues in Acetylcholinesterase which Influence Fasciculin Inhibition

Fasciculin (FAS), a 61 amino acid peptide found in venom of green mambas, is a potent inhibitor of acetylcholinesterases from most species (EC 3.1.1.7)(AChEs) (Karlsson et al., 1984). The crystal structure of FAS (Le Du et al., 1992) reveals close structural features with a larger family of “three finger” snake toxins such as erabutoxin, cardiotoxins, bungarotoxin and cobratoxin. These toxins, however, do not inhibit AChE while FAS at low concentrations does not bind to acetylcholine receptors (Endo and Tamiya, 1987; Karlsson et al., 1984).

Amino Acid Residues that Control Mono- and Bisquaternary Oxime-Induced Reactivation of O-Ethyl Methylphosphonylated Cholinesterases

The limited scope of antidotal activity of commonly used reactivators of organophosphonyl (OP) conjugates of cholinesterases (ChE) such as the methanesulfonate salt of 2-PAM (P2S; Fig. 1) or TMB4, prompted the evaluation of new series of oxime reactivators (Erdman, 1969). This effort resulted in the introduction of a new series of bispyridinium monooximes. One such compound, HI-6 (Fig. 1), is among the most potent reactivating agents that serve as antidotes against organophosphate toxicity (Oldiges and Schoene, 1970).

London Dispersion Interactions in Molecular Recognition by Acetylcholinesterase

Interaction of the aromatic residues W84, Y130 and F330 (or Y330) of Torpedo californica (Nair et al., 1994) and mouse AChEs with the quaternary ammonium function of ligands and substrates has been probed by quantitative structure-activity relationship (QSAR) and site-directed mutagenesis studies. For a series of ten meta-substituted aryl trifluoroketone inhibitors, m-XC6H4COCF3 (X = H, Me, CF3, Et, iPr, tBu, NO2, NH2, NMe2, Me3N+), inhibitor potency (i. e. pKi) is not correlated with substituent hydrophobicity, but is well described by a three-dimensional correlation with the molar refractivity (MR) and σw of the substituents. MR depends on surface area and polarizability, and thus is a measure of London dispersion and other induced polarization interactions between ligands and the aromatic residues of the quaternary ammonium binding locus of the active site. Of the 107-fold range of inhibitor potency, 105 arises from the MR sensitivity, and the corresponding linear subcorrelation indicates that all substituents share a common binding locus and interaction mechanism. A reasonable linear correlation of pKi for inhibition by the m-Me3N+ ketone versus amino acid MR values for a series of W84 mutants of mouse AChE, which includes W84Y, W84F and W84A, indicates that about a third of the binding free energy in the quaternary ammonium binding locus comes from interaction with the tryptophan indole ring. For the native mouse enzyme and these three mutants, a plot of pKi for inhibition by the m-Me3C ketone versus pKi for inhibition by the m-Me3N+ ketone is linear with a slope of 0.7. The greater sensitivity of the charged inhibitor indicates that additional interactions, not enjoyed by the neutral inhibitor, with W84 are operating. These likely include ion-quad-rupole and ion-polarizability interactions with the it-electrons of W84 (Kim et al., 1994). Similar correlations for substrate turnover support the importance of dispersion interactions in AChE catalysis. These studies indicate that the aromatic residues in the quaternary ammonium binding locus of the active site do not function as classical anionic sites.