Zinc enzymes oxidoreductases

This review will give a subjective account of mechanistic studies on some representative zinc enzymes comprising the enzyme classes I-IV (oxidoreductases, transferases, hydrolases and lyases). It does not claim to be comprehensive, as a comprehensive review would be far too extensive for this work. Lowther and Matthews have reviewed the met-alloaminopeptidases and Noodleman and coworkers have reviewed calculational studies on metalloenzymes. We apologize in advance for any omissions and point to previous reviews on this and related subjects to be found, e.g., in References 7-11. 

We have already seen the diversity of function in the lyases, hydrolases and oxidoreductases. Several other types of zinc coordination are found in a number of other enzymes, illustrated in Figure 12.8. These include enzymes with the coordination motif [(His)2(Cys) Zn2+-OH2], illustrated by the lysozyme of bacteriophage T7 this group also includes a peptidyl deformylase. 

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. 

Zinc is an essential trace element. More than 300 enzymes that require zinc ions for activity are known. Most catalyze hydrolysis reactions, but zinc-containing representatives of aU enzyme classes are known, such as, for instance, alcohol dehydrogenase (an oxidoreductase), famesyl-Zgeranyl transferase (a transferase), -lactamase (a hydrolase), carbonic anhydrase (a lyase) and phosphomannose isomerase. 

We have already seen a number of models for the zinc(II) containing enzymes such as carbonic anhydrase in Section 11.3.2. Zinc is an essential component in biochemistry, and forms part of the active site of more then 100 enzymes, of which hydrolases (such as alkaline phosphatase and carboxypeptidase A), transferases (e.g. DNA and RNA polymerase), oxidoreductases (e.g. alcohol dehydrogenase and superoxide dismutase) and lysases (carbonic anhydrase) are the most common. In addition, the non-enzyme zinc finger proteins have an important regulatory function. In many of these systems, the non-redox-active Zn2+ ion is present as a Fewis acidic centre at which substrates are coordinated, polarised and hence activated. Other roles of zinc include acting as a template and playing a structural or regulatory role. 

Metalloenzymes contain a bound metal ion as part of their structure. This ion can either partidpate directly in the catalysis, or stabilize the active conformation of the enzyme. In Lewis acid catalysis (typically with zinc, vanadium, and magnesium), the M"+ ion is used instead of H+. Many oxidoreductases use metal centers such as V, Mo, Co, and Fe in much the same way as homogeneous catalysis uses ligand-metal complexes. Figure 5.7 shows a simplified mechanism for the halide oxidation readion catalyzed by vanadium chloroperoxidase. The vanadium atom ads as a Lewis add, activating the bound peroxide [30]. 

We have already seen the diversity of function in the lyases, hydrolases, and oxidoreductases. Several other types of zinc coordination are found in a number of other enzymes, illustrated in Figure 12.12. These include enzymes with the coordination motif [(His)2(Cys) Zn " -OH2], found in the lysozyme of bacteriophage T7, or [(Cys)3 Zn " "-OH2] which occurs in 5-aminolaevulinate dehydratase (or porphobilinogen synthase). This latter enzyme catalyses the condensation of two molecules of 5-aminolaevulinate to form the pyrrole precursor of the porphyrins (haem, chlorophyll, and cobalamines), and its inhibition by Pb is the cause of lead poisoning (saturnism), frequently observed among inner city children (Chapter 1). 

Assay Methods. 2-Oxoacid ferredoxin oxidoreductase activity is determined by following the absorbance at 550 nm, due to the ferredoxin-dependent reduction of horse heart cytochrome c (Sigma Chemicals, St. Louis, MO) in the presence of 2-oxoacid substrates, essentially as described by Kerscher et al. The assay is conducted at 50°, in 10 mAf potassium phosphate buffer, pH 6.8, in the presence of 2-4 mAf 2-oxoacids (2-oxoglutarate purchased from Nacalai Tesque, Japan, was mainly used), 50-100 p,Af coenzyme A (Kohjin, Japan), 17 pg of the Sulfolobus zinc-containing ferredoxin (purified as described above), 50 pAf horse heart cytochrome c (Sigma Chemicals), and an appropriate amount of enzyme, in a total volume of 1 ml. The reaction is initiated by addition of the enzyme, and nonenzymatic reduction of cytochrome c by coenzyme A at this temperature is... 

In anaerobic archaea, ferredoxin functions as an intermediate electron acceptor of a variety of key steps in the central metabolic pathways involved in saccharolytic and peptide fermentation, and reduced ferredoxin thus formed donates its reducing equivalent to ferredoxiniNADP" oxidoreductase and hydrogenase. - In aerobic and thermoacidophilic archaea, the reoxidation steps of reduced zinc-containing ferredoxin are poorly characterized. The soluble fraction of Sulfolobus sp. strain 7 also contains an NADPH ferredoxin oxidoreductase activity, but this enzyme has not been purified and characterized. The following section describes the purification and partial characterization of a red iron-sulfur flavoprotein with a weak ferredoxin-reoxidizing activity fi om Sulfolobus sp. strain 7. ... 

More than 300 reactions are catalyzed by zinc-containing enzymes [82]. Zinc can be found with insulin, in the reproductive tract, in the DNA-binding proteins, and in oxidoreductases, transferases, lyases, isomerases, and ligases. 

One of the main biochemical roles of zinc is its influence on the activity of over 300 enzymes, which are distributed into the six classes oxidoreductases, transferases, hydrolases, lyases, isomerases, and ligases. 

In order to screen mutants with improved direct electron transfer, it is necessary to use an electrochemical screening system. Currently, only a few electrochemical screening methods were described in literature such as the system developed by the Bartlett group used to screen NADH electro-oxidation. This system uses a multichannel potentiostat with sixty electrodes to screen zinc(n) or ruthenium(ii) complexes bearing the redox phenidione as a mediator for NADH oxidation. It allows the complete evaluation of the electrochemical kinetic constants of the mediators and the immobilization procedure. Unfortunately, this system could only be used with a single electrolyte solution for all the electrodes (e.g., when a single reaction condition or enzyme is assayed), and it requires mL-scale reaction volumes. Recently, another system was described which makes it possible to screen bioelectrocatalytic reactions on 96 independent electrodes screen-printed onto a printed-circuit-board. It showed the possibility to screen direct or mediated electron transfer between oxidoreductases and electrode by intermittent pulse amperometry at the pL-scale (Fig. 6). The direct electron transfer assay was validated with laccase and unmodified electrodes.As an example of the mediated electron transfer assay, the 96 carbon electrodes were modified by phenazines to sereen libraries of a formate dehydrogenase obtained by directed evolution. ... 

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