Binding Properties of an Allosteric Activator Site for Aspartase from Hafnia alvei

Aspartase is a textbook example of a highly specific enzyme with no identifiable alternative substrates. The enzyme is specific to aspartate and fumarate, but NH2OH can be substituted for ammonia as a substrate. A variety of divalent metal ions a such as Mg, Mn, Zn and Co activate the reaction. Initial velocity data for the aspartase from Hafnia alvei are consistent with rapid equilibrium ordered addition of Mg prior to aspartate but with completely random release of Mg, NH4 and fumarate. Using an organic solvent perturbation method, a general base and a general acid for the catalysis of Hafnia alvei aspartase were predicted respectively as a histidine and a cysteine residue. The enzyme can be activated or inhibited when a certain substance binds to a certain site distant from its active site. Aspartase possesses an activator site that has the unusual specificity of binding a substrate, L-aspartate. It has been observed that the occupation of this site with aspartate or with certain structural analogues of the substrate at pH 8.0 and above eliminates the reaction time lags that are observed in the amination direction and the nonlinear kinetics seen in the deamination direction. No time lag was observed in the deamination direction under normal conditions since L-aspartate is already present in the activator site. Aspartase exists in a pH dependent equilibrium between two forms. The higher pH form of aspartase is activated by divalent metal ions and substrate analogues, while the lower pH form of aspartase does not require any effectors for catalytic activity. Aspartase has been reported to be specific for its amino acid substrate L-aspartate, showing no activity with D-aspartic acid, crotonic acid, glycine, alanine, phenylalanine, leucine, methylsuccinate, tyrosine, the mono-and diethyl esters of fumarate, glutamine, maleic acid, or glutaconic acid. Inhibition studies have reported that citrate, EDTA and pyrophosphate block the action of aspartase. Monod and Koshland have previously proposed the models to account for cooperative kinetics and the effects of modifiers on allosteric enzymes. With the observation of time dependent changes in enzyme activities, these models have been extended to allow for the possibility of slow binding of substrates or modifiers and /or conformational changes to account for transient kinetics. In our previous investigation, a time lag was observed during the amination reaction of Hafnia alvei aspartase, which occurred before the final steady-state rate was attained. The time lag was shortened at lower pH, but lengthened at higher pH. Also, the time lag was shortened at higher temperature and lengthened at lower temperature. L-aspartate, D-aspartate and α-methyl-DL-aspartate markedly revealed the activating effects. The time lag and nonlinear kinetics in the amination reaction were eliminated when one of these effectors was added in the reaction mixture. These results suggested that these effectors occupy a distant allosteric activator site apart from the active site. In an effort to elucidate this allosteric activator site in aspartase from Hafnia alvei, we have further investigated the allosteric site properties by virtue of the activity and the time lag changes by using many amino acid analogues of L-aspartate. The enzyme used in this investigation was purified according to the method described by yoon et al. The observation of a time lag during the amination of fumaric acid catalyzed by aspartase has led to various chemicals in an attempt to provide an explanation for this phenomenon. The L-glutamate concentration dependence of the time lag is shown in Figure 1. L-glutamate, an acid amino