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Enzyme-substrate intermediate

Kinetic studies involving enzymes can principally be classified into steady and transient state kinetics. In tlie former, tlie enzyme concentration is much lower tlian that of tlie substrate in tlie latter much higher enzyme concentration is used to allow detection of reaction intennediates. In steady state kinetics, the high efficiency of enzymes as a catalyst implies that very low concentrations are adequate to enable reactions to proceed at measurable rates (i.e., reaction times of a few seconds or more). Typical enzyme concentrations are in the range of 10 M to 10 ], while substrate concentrations usually exceed lO M. Consequently, tlie concentrations of enzyme-substrate intermediates are low witli respect to tlie total substrate (reactant) concentrations, even when tlie enzyme is fully saturated. The reaction is considered to be in a steady state after a very short induction period, which greatly simplifies the rate laws. [Pg.833]

However, in most cases enzymes show lower activity in organic media than in water. This behavior has been ascribed to different causes such as diffusional limitations, high saturating substrate concentrations, restricted protein flexibility, low stabilization of the enzyme-substrate intermediate, partial enzyme denaturation by lyophilization that becomes irreversible in anhydrous organic media, and, last but not least, nonoptimal hydration of the biocatalyst [12d]. Numerous methods have been developed to activate enzymes for optimal use in organic media [13]. [Pg.8]

The haloalkane dehalogenase DhlA mechanism takes place in two consecutive Sn2 steps. In the first, the carboxylate moiety of the aspartate Aspl24, acting as a nucleophile on the carbon atom of DCE, displaces chloride anion which leads to formation of the enzyme-substrate intermediate (Equation 11.86). That intermediate is hydrolyzed by water in the subsequent step. The experimentally determined chlorine kinetic isotope effect for 1-chlorobutane, the slow substrate, is k(35Cl)/k(37Cl) = 1.0066 0.0004 and should correspond to the intrinsic isotope effect for the dehalogenation step. While the reported experimental value for DCE hydrolysis is smaller, it becomes practically the same when corrected for the intramolecular chlorine kinetic isotope effect (a consequence of the two identical chlorine labels in DCE). [Pg.385]

A. Enzyme-Substrate Intermediates (Stop-Action Intermediates) 247... [Pg.245]

All these results are encouraging for investigators planning to use X-ray diffraction in mixed solvents at subzero temperatures and the rest of the present article will be devoted to a discussion of methods and preliminary results in this field. The methodology for cryoprotection of protein crystals, its physical-chemical basis, and the specific problems raised by the crystalline state, as well as the devices used to collect data at subzero temperatures, will be described. Limitations and perspectives of the procedure will be discussed critically. First attempts to determine the structure of productive enzyme-substrate intermediates through stop-action pictures will be described, as well as investigations showing that X-ray diffraction at selected normal and subzero temperatures can reveal protein structural dynamics. [Pg.247]

Work in solution is an absolute prerequisite for further studies of enzyme-substrate intermediates in the crystalline state. According to the Arrhenius relationship, k = A exp(—E IRT), which relates the rate constant k to the temperature, reactions normally occurring in the second to minute ranges might be sufficiently decreased in rate at subzero temperatures to permit intermediates to be stabilized, and occasionally purified by column chromatography if reactions are carried out in fluid solvent mixtures. Therefore, the first problem is to find a suitable cryoprotective solvent for the protein in question. [Pg.247]

While it is tempting to explain regulatory and cosolvent effects on the basis of conformational changes favorable or unfavorable to enzyme activity, it is much more difficult to demonstrate the actual involvement, amount, and structural details of such changes. Experimental evidence consists in most cases of bits and pieces provided by techniques such as absorption and fluorescence spectroscopy, circular dichroism, and magnetic circular dichroism. These tools work in solution (and, when desired, at subzero temperatures) to investigate not simply empty enzymes but enzyme—substrate intermediates. However, even with this information, the conformational basis of enzyme activity remains more postulated than demonstrated at the ball and stick level, and in spite of data about the number and sequence of intermediates, definition of their approximate nature, rate constants, and identification of the types of catalysis involved, full explanation of any particular reaction cannot be given and rests on speculative hypothesis. [Pg.275]

PING PONG HALF-REACTIONS. Many enzymes operate by double-displacement mechanisms involving covalent enzyme-substrate intermediates as shown in the following scheme ... [Pg.330]

Here, / is the electron-transfer rate constants within an outer-sphere enzyme-substrate intermediate the stability of which is... [Pg.229]

Recently, the formation of a covalent glycosyl-enzyme intermediate was also shown by Bell and Koshland (17) in another reaction. Evidence was presented that the mechanism of the enzyme, phosphoribosyl-adeno-sine triphosphate pyrophosphate phosphoribosyl transferase, proceeds through a covalent phosphoribosyl-enzyme intermediate. The intermediate has been demonstrated after incubating the enzyme with 14C-5-phosphoribosyl-l-pyrophosphate (PRPP) under native and denaturing conditions. The intermediate also forms from the reverse direction as shown when the enzyme is mixed with its product N- (5-phosphoribosyl-adenosine triphosphate (PR-ATP). These data give evidence for a covalent enzyme-substrate intermediate. The enzyme which catalyzes the overall reaction proceeds as follows ... [Pg.374]

The structure of the complex of urease with urea in the active site is unknown, because the enzyme-substrate intermediate is very short-lived and has not been trapped. Nevertheless, a number of inhibitors of urease that bridge between the nickel atoms are known. Acetohydroxamate is the most studied and binds slowly but with high affinity (K = 4 vaM [25]). Phosphoroamide is also a slowly binding inhibitor. 2-Thioethanol causes the appearance of sulfur-to-nickel... [Pg.236]

In a review of the proficiencies of enzymes and how they achieve them, it was claimed that ground-state conformations and transition state stabilization cannot explain the very large efficiencies of enzymes instead, they must proceed, it was concluded, by covalent enzyme-substrate intermediates.73 A riposte to this contentious hypothesis has appeared, claiming that account was not taken of the fact that high enzyme efficiency is determined by the value for the water reaction k0 rather than by the enzymatic rate constant kcat/kM.14... [Pg.68]

The chromophoric pyridoxal phosphate coenzyme provides a useful spectrophotometric probe of catalytic events and of conformational changes that occur at the pyridoxal phosphate site of the P subunit and of the aiPi complex. Tryptophan synthase belongs to a class of pyridoxal phosphate enzymes that catalyze /3-replacement and / -elimination reactions.3 The reactions proceed through a series of pyridoxal phosphate-substrate intermediates (Fig. 7.6) that have characteristic spectral properties. Steady-state and rapid kinetic studies of the P subunit and of the aiPi complex in solution have demonstrated the formation and disappearance of these intermediates.73-90 Fig. 7.7 illustrates the use of rapid-scanning stopped-flow UV-visible spectroscopy to investigate the effects of single amino acid substitutions in the a subunit on the rate of reactions of L-serine at the active site of the P subunit.89 Formation of enzyme-substrate intermediates has also been observed with the 012P2 complex in the crystalline state.91 ... [Pg.133]

The mechanism by which influenza virus sialidases cleaves the Neu5Ac(a2 3)Gal or Neu5Ac(a2- 6)Gal linkage has been a topic of much interest for many years (e.g., see [71, 72]). Recently, it has been shown by structural analysis [73] that it involves a covalent enzyme-substrate intermediate as has been reported for other sialidases. A proposed mechanism is depicted in Scheme 17.1. [Pg.463]

Km is the Michaelis constant representing the equilibrium enzyme-substrate intermediate, fcr the rate-determining constant, cE0 the initial enzyme concentration, and S the concentration of substrate. The physical significance is that the reaction rate is proportional to the substrate concentration at low values, but tends to maximum at higher values. If initial rate experiments are conducted then the limit of the reaction rate will correspond to a maximum initial rate Vmr, = krcE0. By replacing it into Eq. (15.1) the following kinetic expression is obtained ... [Pg.442]

Physostigmine [fi zoe STIG meen] is an alkaloid (a nitrogenous compound found in plants) and a tertiary amine. It is a substrate for acetylcholinesterase, and forms a relatively stable enzyme-substrate intermediate that reversibly inactivates acetylcholinesterase. The result is potentiation of cholinergic activity throughout the body. [Pg.53]

In studies of catalase, much effort has been directed toward a determination of whether or not hydrogen peroxide could be dissociated from the enzyme-substrate intermediates of catalases and peroxidases. It should be pointed out that catalase, as contrasted with cytochrome oxidase, has been studied only at room temperature, and if any lesson is to be learned from the study of cytochrome oxidase 150), it is that the complexes are most likely to be identified at low temperatures, as precursors of the compounds. In this sense, they are of first importance and not to be ignored in our understanding of the mechanism of enzymic reactions. [Pg.390]

The chemical shifts of this atom were observed as a function of the pH and the presence and absence of substrate or inhibitor molecules. These titration experiments provided additional evidence for the suggested working model of aspartyl proteases and confirmed that HlV-1 PR is a member of this class of enzymes (26). The two aspartyl side-chain carboxyl groups (one from each subunit) act as general base and acid, respectively, thereby leading to the breakdown of the enzyme-substrate intermediate. [Pg.1787]

Conceptual Insights, Enzyme Kinetics. See the section entitled "Pre-Steady-State Kinetics" in Conceptual Insights module to better understand why a "burst" phase at short reaction times implies the existence of an enzyme-substrate intermediate. [Pg.359]

Kuroki, R., Weaver, L. H., and Matthews, B. W. A covalent enzyme-substrate intermediate with saccharide distortion in a mutant T4 lysozyme. Science 262, 2030-2033 (1993). [Pg.819]


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See also in sourсe #XX -- [ Pg.36 , Pg.50 ]




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