Aspartic Acid Analogues Research Articles

Solvent effects on the spectra of retinal Schiff bases--I. models for the bathochromic shift of the chromophore spectrum in visual pigments.

Abstract— Retinylidine Schiff base spectra have been investigated in a variety of solvents that were chosen primarily to mimic possible environments of the chromophore in visual pigments. These studies have identified particular amino acid side chains which can have important influences on visual pigment spectra. Most amino acid side chain analogues or solvents which mimic the peptide bond environment have little effect on retinylidine Schiff base spectra. Tyrosine, tryptophan, and cysteine analogues and ionized aspartic and glutamic acid analogues produce pronounced red shifts in the spectra. As is explained in more detail in the following paper the presence of these amino acids in the chromophore “pocket” of the protein is sufficient to account for the spectra of many visual pigments. Calculations of the spectra of protonated Schiff bases under a variety of conditions have been considered in conjunction with the spectral data to provide a model for the nature of the chromophore interactions with its environment.In solution and in rhodopsin the protonated Schiff base linkage is likely to be associated with an anion. The presence of certain amino acid side chains along the remainder of the retinal chain in the protein can modify the spectrum by altering the polarizability and polarity of the environment. The polarity in the region of the ionone ring appears to be particularly important for shifting the spectrum. In solution the nucleophilicity of the solvent also has an important influence on the anion‐protonated Schiff base region of the chromophore.

Inhibition of urea cycle enzymes by aspartic acid analogues.

The inhibition of urea biosynthesis by analogues of aspartic acid was studied in vitro in homogenates and enzyme preparations from rat liver. Each of the analogues tested inhibited the overall utilization of citrulline for urea formation by liver homogenates. The concentrations required to give 50% inhibition were: N-allylaspartate, 0.248 M; α-methylaspartate, 0.140 M; β-methylaspartate, 0.078 M; and β-hydroxy-β-methylaspartate, 0.038 M. The β-substituted analogues partly replaced aspartate as a substrate for citrulline utilization in liver homogenates. The replacement was probably due to transamination of the analogues with oxaloacetate, since the effect was not observed when the assay mixture did not contain a substrate which could yield oxaloacetate.A study of individual enzymes of the urea cycle showed that arginase, argininosuccinase, and ornithine transcarbamylase were not greatly affected by the analogues. However, carbamyl phosphate synthetase as well as argininosuccinate synthetase were strongly inhibited, suggesting that the analogues act by some mechanism other than simple antagonism of aspartate. Part of the inhibition was related to the ability of the analogues to complex Mg2+, since increased concentrations of Mg2+ prevented the inhibition of carbamyl phosphate synthetase and reduced the inhibition of argininosuccinate synthetase by α-methylaspartate and N-allylaspartate. In addition, β-methylaspartate was found to depress oxidative and phosphorylative reactions, thus interfering with the energy production required for urea formation.Aspartic acid in concentrations comparable with those required to effect inhibition by α-methylaspartate produced a marked inhibition of citrulline utilization in liver homogenates and of purified argininosuccinate synthetase. This observation suggests that part of the inhibitions observed with the analogues are of the "substrate type".

Transport of aminophosphonic acids in Lactobacillus plantarum and Streptococcus faecalis.

Aminophosphonic acids analogous to glutamic acid, aspartic acid, alanine, and valine were actively accumulated by Lactobacillus plantarum. Uptake was dependent on the availability of glucose and, in all cases, the estimated intracellular concentrations substantially exceeded extracellular levels. During uptake, there was little metabolism of tritiated 2-amino-3-phosphonopropionic acid (APP), the aspartic acid analogue, and a negligible incorporation of isotope from this substance into the nucleic acid, lipid, protein, or cell wall fractions of the cell. Competition studies with APP indicated that its transport in L. plantarum and in Streptococcus faecalis was antagonized only by structurally related compounds such as glutamic, aspartic, and cysteic acids. Kinetic studies showed that APP was taken up by a single catalytic system in S. faecalis. A mutant strain of this organism which lacks one of two kinetically distinguishable dicarboxylic amino acid transport systems failed to accumulate measurable amounts of APP. These experiments indicate that the aminophosphonic acids are accumulated by the amino acid transport systems in these bacteria with minimal metabolic changes.

Effects of analogues of aspartic acid on enzymes of urea synthesis

Analogues of aspartic acid have been studied as inhibitors of urea synthesis starting from citrulline and aspartate in homogenates of rat liver and their effects compared with those on the isolated enzymes. The analogue α-methyl- d, l-aspartate behaved as a weak, competitive inhibitor, consistent with expectation based on the K i for argininosuccinate synthetase. The site of inhibition of urea synthesis is thus confined to the synthetase site. The compound threo-β-methyl- l-aspartate is a substrate for the synthetase and gives rise to the methyl analogue of argininosuccinate, arginino-β-methylsuccinate. The latter compound was prepared enzymatically; it acts as inhibitor for argininosuccinase, in competition with argininosuccinate. The weak inhibition of urea synthesis observed in the presence of β-methyl- l-aspartate was consistent with the K m of this analogue for the isolated synthetase. The site of inhibition is consequently confined mainly to the synthetase site and arises from competition between the two substrates. Little inhibition occurs at the argininosuccinase site due to the low level of arginino-β-methylsuccinate formed and to the appreciable steady-state level of argininosuccinate found to be present during urea synthesis. Administration of either α-methyl- or β-methylaspartate to rats by intraperitoneal or intracardiac injection produced no effects on urea synthesis. The effects of the threo and erythro forms of β-hydroxy- d, l-aspartate and β-hydroxy-β-methyl- d, l-aspartate on isolated argininosuccinate synthetase were examined. Only threo-β-hydroxy- d, l-aspartate was active as substrate. The remaining three compounds acted as inhibitors. The relationship of configuration to enzyme affinity is discussed.