Substrate (chemistry)

About: Substrate (chemistry) is a research topic. Over the lifetime, 35902 publications have been published within this topic receiving 740722 citations. The topic is also known as: enzyme substrate.

Carbamoylphosphate synthetase I of rat-liver mitochondria. Purification, properties, and polypeptide molecular weight.

Abstract: An improved purification of carbamoylphosphate synthetase I from rat liver mitochondria is described. The enzyme is essentially homogeneous with enhanced specific activity and stability. The enzyme is stable at alkaline pH in concentrated solution of salts. Kinetic studies indicate two type of inactivation, reversible and irreversible, depending on pH. The monomeric unit of carbamoylphosphate synthetase I consists of one polypeptide chain. The protein migrates in dodecylsulfate/polyaacrylamide gel as a single component corresponding to a molecular weight of about 155000. The molecular weight of the polypeptide chain determined by sedimentation equilibrium in 6 M guanidine hydrochloride containing dithioerythritol is 158000 ± 5000. The weight-average molecular weight of the native enzyme, 188000, suggests that the monomeric form is the predominant form of the enzyme, under the conditions described. Glutamine is inactive as a substrate. With ammonium, carbamoyl phosphate synthesis is optimal in the pH range 6.8–7.6. Mg2+ and Mn2+ are equally effective metal ions in the direction of carbamoyl phosphate synthesis, but excess Mn2+ is inhibitory. At less than saturating concentrations of substrate, considerable activation of the enzyme is observed when metal ions are in excess of ATP; when MgATP2− concentration is held constant at 1 mM and 10 mM, plots of v versus free Mg2+ concentration are hyperbolic, and extrapolate to zero. These results suggests that free divalent metal ion is required in the forward reaction. The apparent Km of the substrates is similar to other preparations of the enzyme. The determination of the maximal activity of carbamoylphosphate synthetase in rat liver mitochondria is reported, providing a measure of the total enzyme concentration that is about 1–1.5 mM. The purification procedure described also provides an easy method to obtain ornithine transcarbamylase in partially purified form.

Creation of a broad-range and highly stereoselective D-amino acid dehydrogenase for the one-step synthesis of D-amino acids.

Abstract: Using both rational and random mutagenesis, we have created the first known broad substrate range, nicotinamide cofactor dependent, and highly stereoselective d-amino acid dehydrogenase. This new enzyme is capable of producing d-amino acids via the reductive amination of the corresponding 2-keto acid with ammonia. This biocatalyst was the result of three rounds of mutagenesis and screening performed on the enzyme meso-diaminopimelate d-dehydrogenase. The first round targeted the active site of the wild-type enzyme and produced mutants that were no longer strictly dependent on the native substrate. The second and third rounds produced mutants that had an increased substrate range including straight- and branched-aliphatic amino acids and aromatic amino acids. The very high selectivity towards the d-enantiomer (95 to > 99% e.e) was shown to be preserved even after the addition of the five mutations found in the three rounds of mutagenesis and screening. This new enzyme could complement and improve upon current methods for d-amino acid synthesis.