Enzymatic proton transfer

Silverman, D.N. (2000). Marcus rate theory applied to enzymatic proton transfer. Bio-chem. Biophys. Acta. 1458 (1), 88-103... 

Large kinetic isotope effects in enzymatic proton transfer and the role of substrate oscillations, Proc. Natl. Acad. Sci. USA 1997, 94, 12360-12365. 

Silverman, D. N., Marcus Rate Theory Applied to Enzymatic Proton Transfer, Biochem. Biophys. Acta. 2000, 1458, (1), 88-103. 

Rather than attempting an exhaustive review of all possible enzymatic proton transfers, which are manifold, we would like to discuss in the following a few well-documented enzymatic mechanisms involving different types of prototropic reactions. Of these, enzymatic amide- and ester-hydrolyses will be treated the most thoroughly, since at the present time they are the most extensively studied and their corresponding non-enzymatic model systems provide the most solid foundation for discussion. These examples will be preceded by a review of experimental techniques often utilized in mechanistic investigations of enzymatic reactions. 

Excellent reviews exist on several important aspects of enzymatic proton transfers. For the enzymology of carbon-acid reactions the reader is referred to the chapter by Rose in The Enzymes [4] an interesting discussion of proton transfers in biological redox reactions can be found in Hamilton s review [5]. 

The methods generally applied to the detection, identification and the mechanistic study of enzymatic proton transfers are essentially the same as the ones developed for the study of organic reactions. Their use for the study of enzymatic processes is not without difficulties, however, and adaptations are often required to contend with the... 

The most important piece of evidence for proton transfer in enzymatic systems comes from the application of deuterium oxide kinetic solvent isotope effects. If the enzymatic rate-limiting step involves a proton transfer in the transition state, a solvent isotope effect ( hjoAdzo) of 2 to 3 is observed, in good agreement with values observed in non-enzymatic proton transfer reactions [13]. The... 

Finally, the use of isotopes in carbon-acid substrates is an invaluable tool for the determination of the stereochemistry of the enzymatic proton transfer. In contrast to organic reactions, stereospecific proton transfers are the rule, rather than the exception, in enzymatic reactions, owing to the inherently asymmetric nature of the protein surface. An example is the pair of isotopic exchange reactions between dihydroxyacetone phosphate and tritiated water catalysed by the enzymes aldolase and triose phosphate isomerase [17]. In the two cases a different a-hydrogen of the ketone is exchanged with water, leading to the two discrete monotritiated derivatives 1 (labelled by the isomerase) and 2 (labelled by the aldolase) ... 

On the basis of presently available information, mandelic acid racemase might display the simplest enzymatic proton transfer mechanism. The enzyme, isolated from Pseudomonas putida, catalyses the epimerization reaction [49] ... 

Much remains to be done before a clear, quantitative mechanistic picture will be available to describe enzymatic proton transfers. From the methodological point of view, the development of models for intramolecular proton transfers appear to be necessary for the quantification of the influence of H-bond strength and bond orienta-... 

Edsall, J. T. George Scatchard, John G. Kirkwood, and the electrical interactions of amino acids and proteins. Trends Biochem. Sci. 7 (1982) 414-416. Eigen, M. Proton transfer, acid-base catalysis, and enzymatic hydrolysis. Angew. Chem. Int. Ed. Engl. 3 (1964) 1-19. 

There are obviously many reactions that are too fast to investigate by ordinary mixing techniques. Some important examples are proton transfers, enzymatic reactions, and noncovalent complex formation. Prior to the second half of the 20th century, these reactions were referred to as instantaneous because their kinetics could not be studied. It is now possible to measure the rates of such reactions. In Section 4.1 we will find that the fastest reactions have half-lives of the order 10 s, so the fast reaction regime encompasses a much wider range of rates than does the conventional study of kinetics. 

Eigen, M., 1964. Proton transfer, acid-base catalysis, and enzymatic hydrolysis. Angewandte Chemie, Int. Ed. 3 1—72. 

The class of proton transfer (PT) reactions plays a major role in many biological processes, including various enzymatic reactions. This class of reactions will be served here as a general example and an introduction for more complicated reactions. As a specific demonstration let s consider a proton transfer between Cys 25 and His 159 in papain. This reaction can be formally described as... 

The optically active Schiff bases containing intramolecular hydrogen bonds are of major interest because of their use as ligands for complexes employed as catalysts in enantioselective reactions or model compounds in studies of enzymatic reactions. In the studies of intramolecularly hydrogen bonded Schiff bases, the NMR spectroscopy is widely used and allows detection of the presence of proton transfer equilibrium and determination of the mole fraction of tautomers [21]. Literature gives a few names of tautomers in equilibrium. The OH-tautomer has been also known as OH-, enol- or imine-form, while NH tautomer as NH-, keto-, enamine-, or proton-transferred form. More detail information concerning the application of NMR spectroscopy for investigation of proton transfer equilibrium in Schiff bases is presented in reviews.42-44... 

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]. 

Protons are in general indispensable for the dismutation of superoxide (Eq. (4)). Also in the case of its dismutation catalyzed by a metal center, two protons are needed for the dissociation of the product (H2O2) from the metal center (Scheme 9). Therefore, a complex which can accept two protons upon reduction and release them upon oxidation is an excellent candidate for SOD activity. The studies on proton-coupled electron transfer in Fe- and Mn-SODs 48), demonstrated that the active site of MnSOD consists of more than one proton acceptor (Scheme 10). Since the assignment of species involved in proton transfer is extremely difficult in the case of enzymatic systems, relevant investigations on adequate model complexes could be of vast importance. H2dapsox coordinates to Fe(II) in its neutral form, whereas in the case of Fe(III) it coordinates in the dapsox form. Thus, oxidation and reduction of its iron complex is a proton-coupled electron transfer process 46), which as an energetically favorable... 

Acid-base catalysis appears to be an important factor in virtually all enzymatic reactions. The rates of proton transfer reactions have been well studied in model systems,30 but not during the course of enzyme catalysis. The protonation and deprotonation of acids and bases can be represented as... 

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