Enzyme kinetics study

Obach, R.S. (1996) The importance of nonspecific binding in in vitro matrices, its impact on enzyme kinetic studies of drug metabolism reactions, and implications for in vitro-in vivo correlations. Drug Metabolism and Disposition, 24 (10), 1047—1049. 

A number of criteria are crucial in the selection of an appropriate buffer to be utilized in an enzyme-kinetic study. Included in these criteria are ... 

A substance (1,2,3-propanetriol HOCH2CH(OH) CH2OH) that is a component and metabolite of many lipids and is commonly used in enzyme kinetic studies, serving as a cryoprotectant and stabilizing agent for pro-teins Glycerol has also been utilized as a cryosolvent in a number of cryoenzymology studies e.g., catalase. ... 

Enzyme kinetic studies of inhibitor are very important for considering as a therapeutic agent. It is interesting to note that isoprenoid-substituted flavonoids having non-steroidal structures are potent un-competitive inhibitors of 5a-reductase. So, it would be expected that the isoprenoid-substituted flavonoid derivative would be an interesting lead compounds for testosterone 5a-reductase inhibitor. 

Two principal methods are widely used for the assay of DPOs. For enzyme kinetic studies, the most appropriate methods are polarographic and use an 02 electrode (Basic Protocol), which allows the direct measurement of the rate of utilization of oxygen and a true comparison of different phenolic substrates. Minor disadvantages of this method are that it requires more specialized equipment and that assays can only be carried out one at a time. Nevertheless it has proven to be the basis of some excellent undeigraduate biochemical laboratory experiments. 

Included in the following table are some data points from a hypothetical enzyme kinetics study. Using a spreadsheet program with graphing abilities (such as Excel), generate a Line-weaver-Burk plot of the data points in the table. Determine the best-fit line for the data along with Vnax, ATm, and r2 (the square of the correlation coefficient of the line). Does this enzyme follow Michaelis-Menten kinetics Why or why not ... 

Yoshida et al. (1997) achieved the production of vanillin by oxidation of vanillylamine using amine oxidase (AO) from Aspergillus niger and monoamine oxidase (MAO) from Escherichia coli. Enzyme kinetic studies have revealed that AO is... 

Pennise and coworkers (2002) described a continuous fluorometric assay for this enzyme for the purpose of high-throughput screens and enzyme kinetics studies. They designed an acceptor... 

Thus, as the first approach to kinetic studies of the action of /8-D-glu-cosiduronase toward synthetically prepared 1-O-acyl-a- and -/3-D-glu-copyranuronic acids, a reliable method that would permit monitoring of the rate of enzymic hydrolysis was needed. For this purpose, To-masic and Keglevic268 developed an analytical procedure, involving the colorimetric reaction of D-glucuronic acid with benzidine in the presence of acetic acid,269 that proved to be fully applicable to enzymic, kinetic studies performed with 1-esters of D-glucuronic acids and with D-glucosiduronic acids as the substrates. 

Takanashi K, Tainaka H, Kobayashi K, et al. CYP2C9 Ile359 and Leu359 variants enzyme kinetic study with seven substrates. Pharmacogenetics 2000 10(2) 95-104. 

Koketsu et al. (2002) reported the DOPAoxidase activities of the 1,3-selenazol-4-one derivatives 19-24 against mushroom tyrosinase [46]. All of these compounds exhibited 33.4-62.1% inhibition of DOPAoxidase activity at a concentration of 500 p,M. Their inhibitory effects were higher than that of ko-jic acid (31.7%) [46]. 2-(4-Methylphenyl)-l,3-selenazol-4-one (19) exhibited the most potent inhibitory effect among them in a dose-dependent manner. Enzyme kinetic studies showed that compound 19 showed competitive in-... 

Most enzyme kinetic studies assume that proton transfer is fast compared with catalysis, but this is also not necessarily so. It has long been known that bimolecular rate constants for proton transfer between electronegative atoms follow an Eigen curve , with the rate in the thermodynamically favourable direction being dilfusion controlled (10 ° M s at ambient temperature) and in the thermodynamically unfavourable direction being s ... 

Affinity how well a drug and a receptor recognize each other. Notice the analogy to the Km value used in enzyme kinetic studies. 

Efficacy the maximal effect an agonist can achieve at the highest practical concentration. Notice the analogy with die VTOX used in enzyme kinetic studies. 

Saraswat, I.D. et al., High-throughput method for enzyme kinetic studies, J. Biomol. Screening, 8, 544, 2003. 

In mice fed a tryptophan-deficient diet ad libitum for 1 week, Jones et al.31 reported on tissue serotonin synthesis rates, systemic tryptophan metabolism, and its response to steroid or cycloheximide treatment. In the experimental mice, brain serotonin synthesis was decreased while duodenal serotonin synthesis was increased following a tryptophan load. Liver total protein was depressed in experimental mice but increased following a tryptophan load. Blood tryptophan (total and free) and albumin were decreased in experimental mice, but ratios of albumin-bound tryptophan were increased. Enzyme kinetic studies indicated that, in experimental mice, brain tryptophan-5-hydroxylase had a reduced Vmax but the enzyme response to tryptophan or hydrocortisone injection was increased. However, hepatic tryptophan-2,3-dioxygenase response to tryptophan or hydrocortisone injection was blunted in experimental mice. 

Previous theoretical and enzyme kinetic studies have identified a number of factors that could be important in activating the NAD(P) cofactor. Several of these factors can be studied by spectroscopy to determine if and ho v the cofactor NAD(P) is activated within the Michaelis complex to facilitate the hydride transfer. 

Both molybdenum hydroxylases can oxidize uncharged and cationic substrates, although at physiological pH aldehyde oxidase is much more active towards the latter group of compounds. It does not necessarily follow, however, that when compounds have a pKa value around 7, the neutral and protonated forms both bind to the enzyme. Kinetic studies at different pH values have indicated that it is the unprotonated form of 2-aminophthalazine that reacts with the molybdenum hydroxylases, either as a substrate for aldehyde oxidase or a competitive inhibitor of xanthine oxidase [211]. In contrast, at pH 7-8 many of the purine and pteridine substrates are present as mixtures of uncharged molecules and monoanions, both of which are rapidly oxidized by xanthine oxidase [181, 200, 206, 210, 212]. 

Potency the quantity of drug required to achieve a desired effect. In D-R measurements, the chosen effect is usually 50% of the maximal effect. The primary determinant is the affinity of the drug for the receptor. Notice the analogy to the Km value used in enzyme kinetic studies. 

Takanashi, K., H. Tainaka, K. Kobayashi, T. Yasumori, M. Hosakawa, and K. Chiba (2000). CYP2C9 Ile and Leu variants Enzyme kinetic study with seven substrates. Pharmacogenetics 10, 95-104. 

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