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.

Substrate binding to histone deacetylases as shown by the crystal structure of the HDAC8-substrate complex.

Abstract: Histone deacetylases (HDACs)—an enzyme family that deacetylates histones and non-histone proteins—are implicated in human diseases such as cancer, and the first-generation of HDAC inhibitors are now in clinical trials. Here, we report the 2.0 Å resolution crystal structure of a catalytically inactive HDAC8 active-site mutant, Tyr306Phe, bound to an acetylated peptidic substrate. The structure clarifies the role of active-site residues in the deacetylation reaction and substrate recognition. Notably, the structure shows the unexpected role of a conserved residue at the active-site rim, Asp 101, in positioning the substrate by directly interacting with the peptidic backbone and imposing a constrained cis-conformation. A similar interaction is observed in a new hydroxamate inhibitor–HDAC8 structure that we also solved. The crucial role of Asp 101 in substrate and inhibitor recognition was confirmed by activity and binding assays of wild-type HDAC8 and Asp101Ala, Tyr306Phe and Asp101Ala/Tyr306Phe mutants.

Crystal structures of medium-chain acyl-CoA dehydrogenase from pig liver mitochondria with and without substrate

Abstract: The three-dimensional structure of medium-chain acyl-CoA dehydrogenase from pig mitochondria in the native form and that of a complex of the enzyme and a substrate (product) have been solved and refined by x-ray crystallographic methods at 2.4-A resolution to R factors of 0.172 and 0.173, respectively. The overall polypeptide folding and the quaternary structure of the tetramer are essentially unchanged upon binding of the ligand, octanoyl (octenoyl)-CoA. The ligand binds to the enzyme at the rectus (re) face of the FAD in the crevice between the two alpha-helix domains and the beta-sheet domain of the enzyme. The fatty acyl chain of the thioester substrate is buried inside of the polypeptide and the 3'-AMP moiety is close to the surface of the tetrameric enzyme molecule. The alkyl chain displaces the tightly bound water molecules found in the native enzyme and the carbonyl oxygen of the thioester interacts with the ribityl 2'-hydroxyl group of the FAD and the main-chain carbonyl oxygen of Glu-376. The C alpha--C beta of the fatty acyl moiety lies between the flavin and the gamma-carboxylate of Glu-376, supporting the role of Glu-376 as the base that abstracts the alpha proton in the alpha--beta dehydrogenation reaction catalyzed by the enzyme. Trp-166 and Met-165 are located at the sinister (si) side of the flavin ring at the surface of the enzyme, suggesting that they might be involved in the interactions with electron transferring flavoprotein. Lys-304, the prevalent mutation site found in patients with medium-chain acyl-CoA dehydrogenase deficiency, is located approximately 20 A away from the active site of the enzyme.

Revised definition of substrate binding sites of papain-like cysteine proteases.

Abstract: A review of kinetic and structural data has enabled us to reconsider the definition of substrate binding sites in papain-like cysteine proteases. Only three substrate binding sites, S2, S1 and S1', involve main as well as side chain contacts between substrate and enzyme residues. Interactions between the enzymes and the substrate P3 and P2' residues are based on side chains (an exception is cathepsin B which is a carboxydipeptidase), so their interaction surface spreads over a relatively wide area. The location and definition of substrate binding sites beyond S3 and S2' is even more questionable.

Separation-free sandwich enzyme immunoassays using microporous gold electrodes and self-assembled monolayer/immobilized capture antibodies

Abstract: A novel separation-free sandwich-type enzyme immunoassay for proteins is performed by designing an electrochemical detection system that enables preferential measurement of surface-bound enzyme-labeled antibody relative to the excess enzyme-labeled reagent in the bulk sample solution. In this initial model system, the assay is carried out using gold-coated microporous nylon membranes (pore size 0.2 micron) which are mounted between two chambers of a diffusion cell. The membrane serves as both a solid phase for the sandwich assay and the working electrode in the three-electrode amperometric detection system. The capture monoclonal antibody is immobilized covalently on the gold side of the membrane via a self-assembled monolayer of thioctic acid. In the separation-free sandwich assay, both model analyte protein (human chorionic gonadotropin; hCG) and alkaline phosphatase labeled anti-hCG (ALP-Ab) are incubated simultaneously with the immobilized capture anti-hCG antibody. Surface-bound ALP-Ab is spatially resolved from the excess conjugate in the bulk sample solution by introducing the enzyme substrate (4-aminophenyl phosphate) through the back side of the porous membrane. The substrate diffuses rapidly through the porous membrane where it first encounters bound ALP-Ab at the gold surface. The enzymatically generated product, aminophenol, is detected immediately by oxidation at the gold electrode (at +0.19 V vs Ag/AgCl), and the magnitude of current is directly proportional to the concentration of hCG in the sample. The response time after substrate addition is less than 1 min, although maximum response toward the analyte protein requires a sample/conjugate preincubation time of 30 min with the porous electrode.(ABSTRACT TRUNCATED AT 250 WORDS)