Enzyme rate effects

To investigate the cofactor requirement and the characteristics of the enzyme, the effects of additives were examined using phenylmalonic acid as the representative substrate. The addition of ATP or ADP to the enzyme reaction mixtures, with or without coenzyme A, did not enhance the rate of reaction. From these results, it is concluded that these co-factors are not necessary for this decarboxylase. It is well estabhshed that avidin is a potent inhibitor of the bio-tin-enzyme complex [11 -14]. In the present case, addition of avidin has no influence on the decarboxylase activity, indicating that the AMDase is not a biotin enzyme. Thus, the co-factor requirements of AMDase are entirely different from those of known analogous enzymes, such as acyl-CoA carboxylases [15], methyhnalonyl-CoA decarboxylases [11] and transcarboxylases [15,16]. 

DERIVATION OF MORE COMPLICATED RATE EQUATIONS. So far, the rate equations that describe one-substrate enzyme systems have been fairly simple, and the usual algebraic manipulations of substitution and/or addition of simultaneous equations have permitted us to obtain the pertinent rate law. When the number of steps increases and especially when there are branched pathways involved, these manual methods become cumbersome, and more systematic procedures are required. The next two sections should allow the reader to develop a working knowledge of effective methods for obtaining multisubstrate enzyme rate expressions. 

Nature often exploits large pJQ shifts in enzymes to effect chemical catalysis similarly, we hoped to apply the large shifts in the effective basicities of encapsulated guests to reaction chemistry. Initial studies focused on the hydrolysis of orthoformates, a class of molecules responsible for much ofthe formulation ofthe Bronsted theory of acids almost a century ago [98]. While orthoformates are readily hydrolyzed in acidic solution, they are exceedingly stable in neutral or basic solution [99]. However, in the presence of a catalytic amount of 1 in basic solution, small orthoformates are quickly hydrolyzed to the corresponding formate ester [38]. Addition of NEt4 to the reaction inhibited the catalysis but did not affect the hydrolysis rate measured in the absence of 1. With a limited volume in the cavity of 1, substantial size selectivity was observed in the orthoformate hydrolysis. Orthoformates smaller than tripentyl... 

The flow of triose phosphates into sucrose is regulated by the activity of fructose 1,6-bisphosphatase (FBPase-1) and the enzyme that effectively reverses its action, PPrdependent phosphofructokinase (PP-PFK-1 p. 527). These enzymes are therefore critical points for determining the fate of triose phosphates produced by photosynthesis. Both enzymes are regulated by fructose 2,6-bisphosphate (F2,6BP), which inhibits FBPase-1 and stimulates PP-PFK-1. In vascular plants, the concentration of F2,6BP varies inversely with the rate of photosynthesis (Fig. 20-26). Phosphofructokinase-2,... 

Conversely, if [S] < C Km, (Eq. (2.42)) reduces to v = (k2/Km)[E]o[S]. This means that the active sites on the enzyme are effectively unoccupied. The ratio k2/Km is also known as the enzyme s specificity constant, a measure of the enzyme s affinity for different substrates. Thus, if the same enzyme can catalyze the reaction of two substrates, S and S, the relative rates of these two reactions are compared using (k2/Km)s (k2/Km)s-. Because the specificity constant reflects both affinity and catalytic ability, it is also used for comparing different enzymes. 

A noncompetitive inhibitor binds at a site other than the active site of the enzyme and decreases its catalytic rate by causing a conformational change in the three-dimensional shape of the enzyme. The effect of a noncompetitive inhibitor cannot be overcome at high substrate concentrations. On a Lineweaver-Burk plot a noncompetitive inhibitor can be seen to decrease Vmax but leave Km unchanged. 

Similar results have been reported for polymorphisms at amino acid 55, with the PONl paraoxonase activity in blood serum from 55M (methionine) homozygotes reduced compared to either the 55L (leucine) homozygotes or the LM heterozygotes (Mackness et al, 1997). While it is intuitive that the rate of detoxification of a substrate would be dependent on the expression levels of these endogenous enzymes, the effects of the genetic polymorphisms suggest that catalytic efficiency is an equally important consideration. 

The limit imposed by the rate of diffusion in solution can also be partly overcome by confining substrates and products in the limited volume of a multienzyme complex. Indeed, some series of enzymes are associated into organized assemblies (Section 17.1.9) so that the product of one enzyme is very rapidly found by the next enzyme. In effect, products are channeled from one enzyme to the next, much as in an assembly line. 

The relative rates of hydrolysis of xylo-oligosaccharides by D-xylanase HC II (Ref. 236) suggested that the binding site of the enzyme is effectively filled by a chain of four D-xylose residues, as illustrated, with the catalytic site situated between subsites 1 and 2. Apparently, the same fit can also be achieved by AX4, as it is... 

Hwang et al.131 were the first to calculate the contribution of tunneling and other nuclear quantum effects to enzyme catalysis. Since then, and in particular in the past few years, there has been a significant increase in simulations of QM-nuclear effects in enzyme reactions. The approaches used range from the quantized classical path (QCP) (e.g., Refs. 4,57,136), the centroid path integral approach,137,138 and vibrational TS theory,139 to the molecular dynamics with quantum transition (MDQT) surface hopping method.140 Most studies did not yet examine the reference water reaction, and thus could only evaluate the QM contribution to the enzyme rate constant, rather than the corresponding catalytic effect. However, studies that explored the actual catalytic contributions (e.g., Refs. 4,57,136) concluded that the QM contributions are similar for the reaction in the enzyme and in solution, and thus, do not contribute to catalysis. 

As noted earlier in this chapter, the apparent Km values of immobilized enzymes vary with the thickness of the diffusion layer surrounding the particles. In packed-bed enzyme reactors, the thickness of this layer varies with the mobile phase flow rate. Faster flow rates produce smaller diffusion layers and therefore K m values that more closely approximate the true Km of the enzyme. This effect has also been observed with the ficin-CM-cellulose reactor, and plots of K m against flow rate Q obtained at different mobile phase flow rates are shown in Figure 4.14. 

In the particular case of PPL and HGL, using pure dicaprin films, it was found that around 1.5% of the total amount of injected enzyme was recovered after film aspiration [115, 116]. As judged by this percentage of hpid-bound enzyme, the effective surface molar excesses of orlistat to film-bound hpase, inducing a 50% reduction in catalytic activity, were estimated as 10- to 20-fold with PPL, RGL and HGL. The stoichiometry at the interface can be described as follows one hpase molecule embedded within 105 substrate molecules wiU be inactivated to half its initial rate by the presence of 10 orhstat molecules. 

Since the time enzyme was pretreated with ultrasonic irradiation could influence the reaction rate, effect of pretreatment time on the reaction was therefore investigated. As shown in Table II, the reaction rate increased with increasing pretreatment time up to 30 min. Further increase in the pretreatment time beyond 30 min, however, resulted in little change in reaction rate. So, the optimum pretreatment time was thought to be 30 min. 

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