Metalloenzyme kinetics

Kinetic and mechanistic studies of the reactivity Zn-Oh ( = 1 or 2) species in small molecule analogs of zinc-containing metalloenzymes, 41, 81 Kinetics and spectroscopy of substituted phenylnitrenes, 36, 255 Kinetics, of organic reactions in water and aqueous mixtures, 14, 203 Kinetics, reaction, polarography and, 5, 1... 

Kinetic evidence obtained for intramolecular proton transfer between nickel and coordinated thiolate, in a tetrahedral complex containing the bulky triphos ligand (Pl PCE CE PPh to prevent interference from binuclear p-thiolate species, is important with respect to the mechanisms of action of a number of metalloenzymes, of nickel (cf. urease, Section VII. B.4) and of other metals (289). 

This zinc metalloenzyme [EC 1.1.1.1 and EC 1.1.1.2] catalyzes the reversible oxidation of a broad spectrum of alcohol substrates and reduction of aldehyde substrates, usually with NAD+ as a coenzyme. The yeast and horse liver enzymes are probably the most extensively characterized oxidoreductases with respect to the reaction mechanism. Only one of two zinc ions is catalytically important, and the general mechanistic properties of the yeast and liver enzymes are similar, but not identical. Alcohol dehydrogenase can be regarded as a model enzyme system for the exploration of hydrogen kinetic isotope effects. 

There are two attributes of a-D-mannosidase (EC 3.2.1.24) that receive particular attention in this article, namely, its behavior as a zinc-containing metalloenzyme, and its action on naturally occurring, D-mannose-containing molecules. However, such more-systematic considerations as the kinetics of action and the distribution of the enzyme in Nature are not overlooked. 

This enzyme has been studied extensively by x-ray, kinetic, NMR, optical, circular dichroic, and fluorescence techniques. Thus, many approaches have been used to explore the role of the metal ions in catalysis and of other protein residues in substrate binding and catalysis. The review of this enzyme will serve to point out the information to be gained from using multiple biophysical approaches in understanding metalloenzyme catalysis. 

The possible factors involved in the biological selectivity towards metal ions have been considered by Frausto da Silva and Williams3 and by Kustin et al.4 In terms of thermodynamic selectivity a useful formalism for the uptake of any metal ion from a multimetal system is the quotient A Cm, where Km is a relative stability constant and Cm is the concentration of the metal ion. However, as these authors point out,3 a combination of both thermodynamic and kinetic properties must be considered. An appreciation of kinetic factors is often absent in this field, but must be of prime consideration in chelate exchange reactions and in the final irreversible step of metal ion insertion to form the metalloenzymes. 

Kinetic and mechanistic studies of the reactivity Zn-Ohn (n = 1 or 2) species in small molecule analogs of zinc-containing metalloenzymes, 41, 81... 

Carboxypeptidase A was the first metalloenzyme where the functional requirement of zinc was clearly demonstrated (9, 92). In similarity to carbonic anhydrase, the chelating site can combine with a variety of metal ions (93), but the activation specificity is broader. Some metal ions, Pb2+, Cd2+ and Hg2+, yield only esterase activity but fail to restore the peptidase activity. Of a variety of cations tested, only Cu2+ gives a completely inactive enzyme. In the standard peptidase assay, cobalt carboxypeptidase is the most active metal derivative, while it has about the same esterase activity as the native enzyme ((93, 94), Table 6). Kinetically, the Co(II) enzyme shows the same qualitative features as the native enzyme (95), and the quantitative differences are not restricted to a single kinetic parameter. 

In the oxidative polymerization of phenols catalyzed by Cu complexes, the substrate coordinates to the Cu(II) complex and is then activated. The activated phenol couples in the next step. The Cu complex acts effectively as a catalyst at concentrations of 0.2-2 mol% compared to the substrate. The oxidation proceeds rapidly at room temperature under an air atmosphere to give poly(phenylene ether) in a quantitative yield. The polymerization follows Michaelis-Menten-type kinetics [55]. Enzymatic oxidation of phenols is an important pathway in the biosynthesis of lignin in plants [56] catalyzed by a metalloenzyme. 

This chapter mainly focuses on the reactivity of 02 and its partially reduced forms. Over the past 5 years, oxygen isotope fractionation has been applied to a number of mechanistic problems. The experimental and computational methods developed to examine the relevant oxidation/reduction reactions are initially discussed. The use of oxygen equilibrium isotope effects as structural probes of transition metal 02 adducts will then be presented followed by a discussion of density function theory (DFT) calculations, which have been vital to their interpretation. Following this, studies of kinetic isotope effects upon defined outer-sphere and inner-sphere reactions will be described in the context of an electron transfer theory framework. The final sections will concentrate on implications for the reaction mechanisms of metalloenzymes that react with 02, 02 -, and H202 in order to illustrate the generality of the competitive isotope fractionation method. 

APPLICATIONS OF lsO KINETIC ISOTOPE EFFECTS TO REACTIONS OF METALLOENZYMES... 

Further progress in the experimental and computational methodology is essential to address the following (i) the relationship between kinetic and equilibrium isotope effects, (ii) the roles of excited vibrational states, and (iii) how small molecule activation reactions in metalloenzymes relate to those of synthetic inorganic compounds. Once these issues are better understood, isotope fractionation patterns in complex and natural environments can be interpreted at the molecular level. This level of analysis will advance the utility of isotope fractionation in many types of laboratories especially those concentrating on small molecule reactivity. 

The specificities of the various digestive exo- and endopep-tidases suggest that they act synergistically to fulfill a major nutritional function. The concerted action of trypsin, chy-motrypsin, pepsin, and carboxypeptidases A and B facilitate and ensure formation of essential amino acids. The chemical characteristics and metalloenzyme nature of two bovine exopeptidases, lens aminopeptidase and pancreatic carboxy-peptidase A, indicate similarities in their mechanisms of action. However, the aminopeptidase exhibits an unusual type of metal ion activation not observed unth carboxy-peptidase. Chemical and physicochemical studies reveal that the latter enzyme has different structural conformations in its crystal and solution states. Moreover, various kinetic data indicate that its mode of action toward ester substrates differs from that toward peptide substrates. The active site metal atom of carboxypeptidase figures prominently in these differences. 

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