Kinetic probe substrates

In addition to the TDI experiment, the partition ratio measures the TDI efficiency. Specifically, the partition ratio is the number of inactivation kinetic events (k nact) versus the number of substrate turnover events per unit enzyme (kcat) [161], Thus, the most potent partition ratio is zero. The most common experimental setup for determining the partition ratio is the titration method that increases the inhibitor concentration relative to a known amount of enzyme. After the incubations, a secondary incubation containing a probe substrate similar to the TDI experiment is used to define the remaining activity. For accurate determination of the partition ratio from the titration method, it is assumed that the inhibitor is 100% metabolized ... 

In a collaboration between the Abelson and Hecht labs [56b], a series of noncoded amino acids were introduced into dihydrofolate reductase (DHFR) to probe substrate binding and the requirement of an aspartic acid residue for catalytic competence. When aspartic acid analogs mono- or disubstituted at the )0-carbon were substituted for the active site aspartic acid residue, the mutant DHFRs were still able to catalyze the NADPH-dependent reduction of dihydrofolate to tetrahydrofolate at 74 - 86 % of the wild-type rate. While hydride transfer from NADPH is not the rate-limiting step for the wild-type enzyme at physiological pH, a kinetic isotope experiment with NADPD indicated that hydride transfer had likely become the rate-limiting step for the mutant containing the )0,)0-dimethylaspartic acid. 

There are three basic methods for carrying out alternative substrate inhibition studies. In the first, the investigator seeks to observe numerical changes in the coefficients of the double-reciprocal form of the enzyme rate expression in the presence and absence of the alternative substrate. For some mechanisms, only certain coefficients will be altered. This method requires extremely accurate estimates of the magnitudes of the coefficients and should always be supplemented with other kinetic probes . 

Stereochemical probes of the specificity of substrates, products, and effectors in enzyme-catalyzed reactions, receptor-ligand interactions, nucleic acid-ligand interactions, etc. Most chirality probe studies attempt to address the stereospecificity of the substrates or ligands or even allosteric effectors. However, upon use of specific kinetic probes, isotopic labeling of achiral centers, chronfium-or cobalt-nucleotide complexes, etc., other stereospecific characteristics can be identified, aU of which will assist in the delineation of the kinetic mechanism as well as the active-site topology. A few examples of chirality probes include ... 

Tabrett CA, Coughtrie MWH (2003) Phenol sulfotransferase 1A1 activity in human liver Kinetic properties, interindividual variation and re-evaluation of the suitability of 4-nitrophenol as a probe substrate. Biochem Pharmacol... 

Galetin, A., Brown, C., Hallifax, D., Ito, K. and Houston, J.B. (2004) Utility of recombinant enzyme kinetics in prediction of human clearance impact of variability, CYP3A5, and CYP2C19 on CYP3A4 probe substrates. Drug Metabolism and Disposition The Biological Fate of Chemicals, 32, 1411-1420. 

The use of RAIRS has recently been extended from its regular mid-IR characterization of adsorbates on metals into other exciting and promising directions. For one, changes in optics and detectors have allowed for an extension of the spectral range towards the far-IR region in order to probe substrate-adsorbate vibrations [35]. The use of intense synchrotron sources in particular looks quite promising for the detection of such weak modes [36]. Thanks to the speed with which Fourier-transform spectrometers can acquire complete IR spectra, kinetic studies of surface reactions can be carried out as well. To date this has only been done in a few cases, usually for reactions that take seconds or more to occur [37], but the advent of step scanners promises... 

The kinetic probes 1 and 2 were selected because the hydrolysis of substrates is characterized by pH-independent rates between pH = 2-6. This is also the pH region in which at-PMAA undergoes a compact-coil to random-coil transition. By changing the hydrophobicity of substrates through variation of R, / i and R2 in I and 2, it is also possible to elucidate the role of hydrophobic microdomains inside PMAA. 

Hutzler JM, Balogh LM, Zientek M, Kumar V, Tracy TS (2009) Mechanism-based inactivation of cytochrome P450 2C9 by tienilic acidand (+/-)-su-profen a comparison of kinetics and probe substrate selection. Drug Metab Dispos 37 59-65... 

Acidity of the reaction mixes after incubation increased as the activity of the probe ro —se during determination of pectinesterase activity of the samples.1t was caused by the for—mation of carboxyl groups as a result of pectin ester bonds hydrolysis under pectinesterase ac—tion.That is why kinetic characteristics of substrate hydrolysis were measured according to the speed of pectin hydrolysis by continuously recorded titration of the free carboxyl groups (11). 

AMPPD is the best chemiluminescent substrate for detecting an ALP-labeled probe [2, 3], The enhanced sensitivity of the chemiluminescence based on the reaction of AMPPD with ALP depends on the enzymatic reaction time (Fig. 2), because the slow kinetics of the signal decay result in the accumulation... 

Because solvent viscosity experiments indicated that the rate-determining step in the PLCBc reaction was likely to be a chemical one, deuterium isotope effects were measured to probe whether proton transfer might be occurring in this step. Toward this end, the kinetic parameters for the PLCBc catalyzed hydrolysis of the soluble substrate C6PC were determined in D20, and a normal primary deuterium isotope effect of 1.9 on kcat/Km was observed for the reaction [34]. A primary isotope effect of magnitude of 1.9 is commonly seen in enzymatic reactions in which proton transfer is rate-limiting, although effects of up to 4.0 have been recorded [107-110]. 

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