Steady-state kinetic studies

The mechanism of the first half-reaction has been studied by a combination of reductive titrations with CO and sodium dithionite and pre-steady-state kinetic studies by rapid freeze quench EPR spectroscopy (FQ-EPR) and stopped-flow kinetics 159). These combined studies have led to the following mechanism. The resting enzyme is assumed to have a metal-bound hydroxide nucleophile. Evidence for this species is based on the similarities between the pH dependence of the EPR spectrum of Cluster C and the for the for CO, deter-... 

The determination of bisubstrate reaction mechanism is based on a combination of steady state and, possibly, pre-steady state kinetic studies. This can include determination of apparent substrate cooperativity, as described in Chapter 2, study of product and dead-end inhibiton patterns (Chapter 2), and attempts to identify... 

A steady-state kinetics study for Hod was pursued to establish the substrate binding pattern and product release, using lH-3-hydroxy-4-oxoquinoline as aromatic substrate. The reaction proceeds via a ternary complex, by an ordered-bi-bi-mechanism, in which the first to bind is the aromatic substrate then the 02 molecule, and the first to leave the enzyme-product complex is CO [359], Another related finding concerns that substrate anaerobically bound to the enzyme Qdo can easily be washed off by ultra-filtration [360] and so, the formation of a covalent acyl-enzyme intermediate seems unlikely in the... 

A number of different approaches have been employed in different laboratories to characterize cyt c ccp binding. The earliest estimates of binding constants come from steady state kinetic studies by Yonetani and coworkers [19] (subsequently refined by Erman) [29]. At 50 mM phosphate, pH6, (conditions which favor maximum turnover), an apparent Km value of 3 pM is obtained using yeast isol cyt c as the reaetion partner of ccp. Km is intrinsically a kinetic parameter, which in the complex ccp mechanism may incorporate a number of elementary rate constants unrelated to binding. 

In steady-state kinetic studies, the total concentration of the enzyme should be much less than the concentration of the substrate(s), product(s), and effector(s) typically, by at least a thousandfold. When this condition is not true, the steady-state condition will not be valid and other methods, such as global analysis, have to be utilized to analyze the kinetic data. 

It has been known for some time that two molecules of 6,7-dimethyl-8-D-ribityllumazine dismute to form riboflavin. An intermediate pentacyclic compound has now been detected by pre-steady-state kinetic studies <2003JBC47700>. 

Martinelli, R. A., and Scheraga, H. A. (1980). Steady-state kinetic study of the bovine thrombin-fibrinogen interaction. Biochem. 19, 2343-2350. 

Fiala, K. A., and Suo, Z. (2004b). Pre-steady-state kinetic studies of the fidelity of Sulfolohus solfataricus P2 DNA polymerase IV. Biochemistry 43, 2106-2115. 

The steady-state kinetic studies of liver alcohol dehydrogenase (12.5 nM) are performed. The initial rates (v in /rM/rnin) with varying substrate concentrations in both directions (forward for ethanol oxidation and reverse for ethanal reduction) are given below. Evaluate their kinetic parameters and equilibrium constant. 

Stable Co111 ADP and ATP complexes have been used as competitive inhibitors in a number of enzymic studies and some progress has been made at unravelling the requirements of the active sites. Steady-state kinetic studies show jff,y-[Co(ATP)(NH3)4] to compete with MnATP for (Na+ + K+), Mg2+ and Ca2+ ATPases derived from kidney medulla.574 values for /J,y-[Co(ATP)(NH3)4] are similar to the Km values for MnATP for both the (Na+ + K+) and Mg24- enzymes, and 3,P NMR shows that the Co111 complex acts as a substrate for the Mg2+ and Ca2+ systems. Likewise,... 

Dissociation of NADH prior to phosphorolysis is a feature common to many mechanisms for GAPDH (cf. 13) since this is necessary to explain the NAD+ requirement for phosphorolysis of the acyl enzyme (196). Indeed, apart from the earlier steady-state kinetic studies, the only report at variance with this concept is that of Smith (197), who examined the ternary acyl enzyme-NADH complex fluorimetrically and was unable to displace the NADH even at high concentrations of NAD. It has, however, been suggested (117) that phosphate is required for this displacement to occur. 

Pre-steady state kinetic studies have provided information on the free energy profile of the glutamate mutase reaction (Figure 24) (Chih and Marsh, 1999 Marsh and Ballou, 1998). In contrast to MMCM, rapid quench... 

Steady-state kinetic studies on a number of enzymes support an ordered mechanism in which the a-keto acid binds first. This observation suggests the formation of an iron-a-keto acid adduct to initiate the activation of O2. Consistent with this notion, crystallographic and... 

Steady-state kinetic studies showed that the kinetics of the enzyme resemble those of the vanadium bromoperoxidases. The chloroperoxidase exhibits a pH profile similar to vanadium bromoperoxidases although the optimal pH of 4.5-5.0 is at a lower value. At low pH the enzyme is inhibited by chloride in a competitive way whereas at higher pH values the activity displays normal Michaelis-Menten type of behavior (see Michaelis Constant). The log Km for chloride increases linearly with pH whereas that for hydrogen peroxide decreases with pH demonstrating that in the catalytic mechanism protons are involved. These observations have led to a simplified ping-pong type of mechanism for the chloroperoxidase similar to that shown in (Figure 1). 

Dolence, J.M., Cassidy, P.B., and Mathis, J.R. (1995). Yeast protein farnesyltransferase steady-state kinetic studies of substrate binding. Biochemistry 34 16687-16694. 

Early steady state kinetic studies established techniques for monitoring the overall reaction and for determining substrate specificity. The most generally applicable method for determining steady state rates of the oxidases is O2 consumption. Oxygen electrode techniques (28) have now superseded earlier manometric methods. The enzyme preparations must either be completely free of catalase activity, as a result of high enzyme purity or addition of cyanide, or catalase must be added in amounts sufficient to prevent transient H2O2 accumulation. 

Fee JA, Bull C. (1982) Steady-state kinetic studies of superoxide dismutases. Saturative behavior of the copper- and zinc-containing protein. J Riol Chem 261 13000-13005. 

Traditional steady-state kinetic studies rely on indirect observation of catalysis by monitoring the accumulation of product or consumption of substrate as a consequence of many reaction cycles with a trace of catalyst. Conclusions are limited to inference of the possible pathways for the order of addition of multiple substrates and release of products and quantification of two bulk kinetic parameters, kcat and kcaJKm- The parameter kcat defines the maximum rate of conversion of enzyme-bound substrate to product released into solution, but it cannot be used to establish whether the maximum rate of reaction is limited by enzyme conformational changes, rates of chemical reaction, or rates of product release per se it does, however, set a lower... 

The advantage of the partial oxidation of methanol of supported metal oxides compared to neat oxides has been shown by transient and steady-state kinetic studies (251). Other recent progress in this field has been discussed (252). Another transient study involves various copper catalj sts (255), and particularly noteworthy in this study was the observation of responses to oscillation of the feed composition and the development of self-sustained oscillations. 

Steady-state kinetic studies indicate that Km(GMP) and Ki(CMP) of the mutant increase by 30-fold while kcat of the mutant decreases by 6-fold. kcat/Km(GMP) of the mutant decreases by -230-fold. The mutation causes relatively little changes in other kinetic parameters. The kinetic results are corroborated by substrate titration studies. The titration data indicate that the affinity of Y50F for MgATP is similar to that of the wild-type enzyme, but the affinity of the mutant for GMP decreases dramatically. Since the effects of the mutation on the kinetic and binding properties of the enzyme are rather specific and the structure of the mutant is highly similar to that of the wild-type GK, the kinetic... 

Pre-steady-state kinetic studies established that the appearance of the NADH chromophore on addition of substrate was a two-step process, and these steps can now be identified as closure of the active site and hydride transfer. This study indicated that the on-enzyme equilibrium for addition of water or homocysteine to the enone was close to unity (and the value in free solution), whereas the equilibrium for oxidation of NAD by bound adenosine was 10 times more favourable than in free solution. The focusing of the catalytic power of the enzyme on the oxidation step avoids the formation of abortive complexes by hydride transfer between enone and NADH, yielding 4,5-dehydroadenosine and NAD ". This happens about 10 " times faster than productive hydride transfer at the beginning and end of the catalytic cycle, with the slow rate (close to that of model reactions) apparently arising from a conformationally modulated increase in the distance the hydride has to be transferred. 

EWOD-based digital microfluidics have already been combined with MALDI-TOF-MS by Wheeler et al.13 However, the Wheeler et al. device would have several drawbacks when used in pre-steady state kinetic studies, including lower throughput - for reasons explained in the Section 12.3 - and the absence of a mixing element. Further, while Paik et al.5 have demonstrated droplet mixers for EWOD-based systems, their system is simply not fast enough to be of use in pre-steady state kinetics. 

The main parts of this scheme were proposed earlier by Theorell and co-workers 119,291) on the basis of inhibitor binding and steady-state kinetic studies. Other suggested mechanisms based on general acid-base catalysis 297), reduction of the enzyme 362), or direct participation of histidine 363) or cysteine 364) in the hydride transfer step are highly unlikely in view of the crystallographic and kinetic results reviewed in this chapter. Contrary to expectations the mechanism described here is in most details very different from that proposed for lactic dehydrogenase 126). 

---