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.

Stochastic inhibitor release and binding from single-enzyme molecules

Abstract: Inhibition kinetics of single-beta-galactosidase molecules with the slow-binding inhibitor d-galactal have been characterized by segregating individual enzyme molecules in an array of 50,000 ultra small reaction containers and observing substrate turnover changes with fluorescence microscopy. Inhibited and active states of beta-galactosidase could be clearly distinguished, and the large array size provided very good statistics. With a pre-steady-state experiment, we demonstrated the stochastic character of inhibitor release, which obeys first-order kinetics. Under steady-state conditions, the quantitative detection of substrate turnover changes over long time periods revealed repeated inhibitor binding and release events, which are accompanied by conformational changes of the enzyme's catalytic site. We proved that the rate constants of inhibitor release and binding derived from stochastic changes in the substrate turnover are consistent with bulk-reaction kinetics.

Decarboxylation of fatty acids to terminal alkenes by cytochrome P450 compound I.

Abstract: OleTJE, a cytochrome P450, catalyzes the conversion of fatty acids to terminal alkenes using hydrogen peroxide as a cosubstrate. Analytical studies with an eicosanoic acid substrate show that the enzyme predominantly generates nonadecene and that carbon dioxide is the one carbon coproduct of the reaction. The addition of hydrogen peroxide to a deuterated substrate–enzyme (E–S) complex results in the transient formation of an iron(IV) oxo π cation radical (Compound I) intermediate which is spectroscopically indistinguishable from those that perform oxygen insertion chemistries. A kinetic isotope effect for Compound I decay suggests that it abstracts a substrate hydrogen atom to initiate fatty acid decarboxylation. Together, these results indicate that the initial mechanism for alkene formation, which does not result from oxygen rebound, is similar to that widely suggested for P450 monooxygenation reactions.

The allosteric activation of mammalian alpha-amylase by chloride.

Abstract: α-Amylase from hog pancreas is shown to possess one binding site for Cl− per molecule of enzyme with a dissociation constant of 3 × 10−4 at 25 °C. The chloride anion functions as an activating effector increasing kcat 30-fold towards either starch or p-nitrophenylmaltoside. No change in Km for either substrate is seen. The other monovalent anions Br−, I−, NO2−, NO3−, ClO4−, SCN−, N3− and CNO− can substitute for chloride but their effect on kcat decreases as their anionic radius increases. Chloride binding induces a subtle conformational change in the enzyme reflected by the suppression of the exchange of 26 protons and a 240-fold increase in the amylase-Ca2+ binding constant from 8.3 × 108 to 2 × 1011 M−1. The conformational change is minor since it is not detected by circular dichroism, protein fluorescence or by specific probes attached to the enzyme. The ability of small structural changes to induce large catalytic acceleration is discussed.

Characterization of immobilization of an enzyme in a modified Y zeolite matrix and its application to an amperometric glucose biosensor.

Abstract: A new approach to construct an amperometric biosensor is described. Without using bovine serum albumin−glutaraldehyde, glucose oxidase (GOx) was immobilized on a dealuminized Y zeolite (DAY)-modified platinum electrode to construct a glucose sensor. The large specific surface area of the zeolite substrate resulted in high enzyme loading. The immobilized GOx in this manner was stable and could maintain its high activity for at least 3 months. The interactions between the zeolite and the enzyme were investigated by means of Fourier transform infrared spectra, and the pore distribution and the surface acid property of DAY were preliminarily studied. The results showed that the hydrophilic property and the existing mesopores of DAY played important roles in the enzyme immobilization. This resulting biosensor exhibited good reproducibility and selectivity, owing to the uniform pore structure and unique ion-exchange property of the zeolite. The biosensor responded rapidly to glucose in the linear range from 2.0...