Structural Changes In Active Site Research Articles

Revealing substrate-induced structural changes in active site of human CYP51 in the presence of its physiological substrates

The human sterol 14α-demethylases (CYP51, CYP is an abbreviation for cytochrome P450) catalyze three-step oxidative removal of 14α-methyl group of lanosterol by first forming an alcohol, then an aldehyde, and finally conducting a CC bond cleavage reaction. This present study utilizes a combination of Resonance Raman spectroscopy and Nanodisc technology to probe the active site structure of CYP51 in the presence of its hydroxylase and lyase substrates. Ligand-binding induced partial low-to-high-spin conversion is observed by applying electronic absorption spectroscopy and Resonance Raman (RR) spectroscopy. This low degree of spin conversion of CYP51 is contributed by the retention of the water ligand coordinated to the heme iron as well as direct interaction between the hydroxyl group of lyase substrate and the iron center. No significant changes in active site structure are found between detergent-stabilized CYP51 and nanodisc-incorporated CYP51, nevertheless, it is demonstrated that nanodisc-incorporated assemblies provide much more well-defined active site RR spectroscopic responses, which induces a larger conversion from low-to-high-spin state in presence of the substrates. Moreover, a positive polar environment around the exogenous diatomic ligand is detected, providing insight into the mechanism of this essential CC bond cleavage reaction.

The main factor of the decrease in activity of luciferase on the Si surface

The interactions between the luciferase surface area around the active site and the Si surface were estimated by molecular dynamics simulations. The results show that the luciferase surface area around the active site is more unstable than the luciferase surface area with relatively high hydrophobicity when luciferase adsorbs on the Si surface. The main factor of the decrease in activity of luciferase on the Si surface would be adsorption-induced structural changes in active site. Si surface should be designed focusing on structural changes in active site.

Analysis of structural changes in active site of luciferase adsorbed on nanofabricated hydrophilic Si surface by molecular-dynamics simulations

Interactions between luciferase and a nanofabricated hydrophilic Si surface were explored by molecular-dynamics simulations. The structural changes in the active-site residues, the residues affecting the luciferin binding, and the residues affecting the bioluminescence color were smaller on the nanofabricated hydrophilic Si surface than on both a hydrophobic Si surface and a hydrophilic Si surface. The nanofabrication and wet-treatment techniques are expected to prevent the decrease in activity of luciferase on the Si surface.

Specific and Amplified Current Responses to Histidine and Histamine Using Immobilized Copper–Monoamine Oxidase Membrane Electrode, Based on Novel Ascorbate Oxidase Activity Induced by Exogenous Ligands

Upon the addition of histidine and histamine, the copper containing monoamine oxidase (MAO) catalyzes the oxidation ofl-ascorbic acid (AsA) by dissolved oxygen, in which the consumed oxygen was finally converted to hydrogen peroxide, according to Michaelis–Menten kinetics (Km= 1.97 mm, upon the addition of 5 × 10−5m l-histidine at 35°C and pH 8.5). The amount of oxygen consumption depended on the amount of histidine and histamine added, and specific responses over other amines were observed when the oxygen electrode modified with immobilized MAO membrane was used in the 0.1mTris buffer containing AsA. The calibration curves ofl-histidine and histamine at 4 mmAsA exhibit linearity in the concentration range from 5 × 10−7to 5 × 10−5m l-histidine and 5 × 10−6to 1 × 10−3mhistamine, with detection limit of 3 × 10−7m l-histidine and 3 × 10−6mhistamine, respectively. The ESR signal of copper(II) in active site of MAO at 77K was apparently changed upon the addition ofl-histidine and histamine indicating that exogenous histidine and histamine bound to the copper site of enzyme and lead to the structural change in active site.