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.

The crystal structure of xanthine oxidoreductase during catalysis: Implications for reaction mechanism and enzyme inhibition

Abstract: Molybdenum is widely distributed in biology and is usually found as a mononuclear metal center in the active sites of many enzymes catalyzing oxygen atom transfer. The molybdenum hydroxylases are distinct from other biological systems catalyzing hydroxylation reactions in that the oxygen atom incorporated into the product is derived from water rather than molecular oxygen. Here, we present the crystal structure of the key intermediate in the hydroxylation reaction of xanthine oxidoreductase with a slow substrate, in which the carbon–oxygen bond of the product is formed, yet the product remains complexed to the molybdenum. This intermediate displays a stable broad charge–transfer band at ≈640 nm. The crystal structure of the complex indicates that the catalytically labile Mo—OH oxygen has formed a bond with a carbon atom of the substrate. In addition, the Mo⋕S group of the oxidized enzyme has become protonated to afford Mo—SH on reduction of the molybdenum center. In contrast to previous assignments, we find this last ligand at an equatorial position in the square-pyramidal metal coordination sphere, not the apical position. A water molecule usually seen in the active site of the enzyme is absent in the present structure, which probably accounts for the stability of this intermediate toward ligand displacement by hydroxide.

Over-stabilization of Candida antarctica lipase B by ionic liquids in ester synthesis

Abstract: Four different ionic liquids, based on dialkylimidazolium cations associated with perfluorinated and bis(trifluoromethyl)sulfonyl amide anions were used as reaction media for butyl butyrate synthesis catalyzed by free Candida antarctica lipase B at 2% (v/v) water content and 50 °C. Lipase had enhanced synthetic activity in all ionic liquids in comparison with two organic solvents (hexane, and 1-butanol), the enhanced activity being related to the increase in polarity of ionic liquids. The continuous operation of lipase with all the assayed ionic liquids showed over-stabilization of the enzyme. The reuse of free lipase in 1-butyl-3-methylimidazolium hexafluorophosphate in continuous operation cycles showed a half-life time 2300 times greater than that observed when the enzyme was incubated in the absence of substrate (3.2 h), and a selectivity higher than 90%.

Mechanism of the prolyl hydroxylase reaction. 2. Kinetic analysis of the reaction sequence.

Abstract: The kinetics of the prolyl hydroxylase reaction were studied with pure enzyme from chick embryos by varying the concentration of one substrate in the presence of different fixed concentrations of the second substrate, while the concentrations of the other substrates were held constant. Intersecting lines were obtained in double-reciprocal plots for all possible pairs of Fe2+, 2-oxoglutarate, O2 and the polypeptide substrate, whereas parallel lines were obtained for pairs involving ascorbate with each substrate. In addition, parallel lines were obtained when the polypeptide substrate concentration was varied at different fixed 2-oxoglutarate concentrations in the presence of saturating O2 concentration. Poly(L-proline) was a competitive inhibitor with respect to the polypeptide substrate, but uncompetitive with respect to Fe2+ and 2-oxoglutarate. High concentrations of the polypeptide substrate inhibited the reaction, this substrate inhibition being competitive with respect to Fe2- and 2-oxoglutarate. Succinate, CO2 and collagen were product inhibitors, succinate inhibiting the reaction competitively with respect to 2-oxoglutarate, but noncompetitively with respect to the other substrates, and collagen noncompetitively with respect to all substrates. The apparent Km and Ks values for the substrates and Ki values for the inhibitors are given. These and additional data would be consistent with a tentative reaction scheme involving an ordered binding of Fe2+, 2-oxoglutarate, O2 and the polypeptide substrate to the enzyme in this order, the binding of Fe2+ being at thermodynamic equilibrium. The enzyme can also react directly with the polypeptide substrate or its analogue poly(L-proline) under certain conditions, forming dead-end complexes. The products are released only after the hydroxylation, possibly in the order: the hydroxylated polypeptide, CO2 and succinate. Ascorbate may react either with enzyme·Fe before the release of Fe2+ or with free enzyme before the binding of Fe2+, but a reaction with ascorbate at any stage after the release of the first product is not excluded. The mechanism proposed is not entirely identical with either of the main two previous suggestions for the mechanism of 2-oxoglutarate dioxygenases.

Catalytic activity of mesoporous silicate-immobilized chloroperoxidase

Abstract: A versatile enzyme, FeHeme chloroperoxidase (CPO) from Caldariomyces fumago, is immobilized in the mesoporous silicate material, mesocellular foam (MCF). MCF is a promising material for immobilizing enzymes, due to its large pore structure and high loading capacity compared to other mesoporous materials, such as MCM-48, SBA-16 and SBA-15. The immobilized CPO in MCF retains its activity. The optimal pH at which the maximum amount of enzyme is immobilized was determined to be pH 3.4, slightly below the isoelectric point of the enzyme. A weak ionic interaction between the enzyme and the surface of the inorganic substrate is thought to be critical in maintaining the activity of the immobilized enzyme. The loading capacity of MCF is 122 mg protein per 1 g of MCF. We demonstrate the advantage of MCF as an inorganic substrate for immobilization of enzymes.