Enzymic Reactions involving Zinc

Enzymic Reactions Involving Zinc.—A dipeptidase from a mouse ascites tumour has been shown to require zinc for the catalysed cleavage of alanine-glycine. Substitution with cobalt or manganese alters the kinetic properties of the enzyme and it is similar to other mammalian dipeptidases in other respects as well univalent anions have been found to bind to a renal zinc dipeptidase in a 1 1 mole ratio, a group with a 

Cusvimano, G. Guglielmo, V. Ricento, R. Romeo, and M. Trozzi, Inorg. Chim. Acta, 1976, 17, 45. 

Aylett, The Chemistry of Zinc, Cadmium and Mercury , Pergamon, NY, 1975. 

coli membranes contains zinc, which is required for full activity. A functional role for zinc in protein synthesis elongation factor 1 from rat liver has been inferred from the observation that 0.3 mM 1,10-phenanthroline completely abolishes guano-sine 5 -triphosphate binding to EFl there is one zinc per molecule (54 000 Daltons).  

This section is therefore divided into three parts enzymic reactions involving zinc metal-protein complex formation and studies involving reaction mechanisms. 

The regeneration of the activity of the enzyme by cobalt differs from the behaviour of an alkaline phosphatase isolated from human placenta.228 This enzyme is reported to be re-activated by replacement of the native zinc ion by either zinc, magnesium, or mercury. No other metal is active. 

Some insight into the function of the zinc ion in alkaline phosphatase from E. coli has resulted from 35C1 n.m.r. studies.229 The uncertainty which has surrounded the number of zinc ions required for activity of the enzyme has been resolved somewhat by the observation that alkaline phosphatase prepared in the absence of edta requires only 2 moles of Zn2+ per mole of enzyme for full activity. 35C1 line-broadening by n.m.r. shows that on addition of two moles of zinc per mole enzyme, no broadening 

The first neutral zinc-containing endopeptidase has been reported.230 Isolated from the brush-border of rabbit kidney, it has a molecular weight of ca. 93 000 and 1 atom of Zn2+ per mole. It is inhibited by edta and reactivated by zinc and other bivalent metals, less zinc being required for full reactivation. 

Garner and Behai have reported the isolation of a zinc-activated aminopeptidase from human liver.231 The enzyme, which is capable of forming a cobalt-enzyme complex, has zinc located at or near the substrate binding site. 

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One of the most important metals with regard to its role in enzyme chemistry is zinc. There are several significant enzymes that contain the metal, among which are carboxypeptidase A and B, alkaline phosphatase, alcohol dehydrogenase, aldolase, and carbonic anhydrase. Although most of these enzymes are involved in catalyzing biochemical reactions, carbonic anhydrase is involved in a process that is inorganic in nature. That reaction can be shown as... 

The next five transition metals iron, cobalt, nickel, copper and zinc are of undisputed importance in the living world, as we know it. The multiple roles that iron can play will be presented in more detail later in Chapter 13, but we can already point out that, with very few exceptions, iron is essential for almost all living organisms, most probably because of its role in forming the amino acid radicals required for the conversion of ribonucleotides to deoxyribonucleotides in the Fe-dependent ribonucleotide reductases. In those organisms, such as Lactobacilli6, which do not have access to iron, their ribonucleotide reductases use a cobalt-based cofactor, related to vitamin B12. Cobalt is also used in a number of other enzymes, some of which catalyse complex isomerization reactions. Like cobalt, nickel appears to be much more extensively utilized by anaerobic bacteria, in reactions involving chemicals such as CH4, CO and H2, the metabolism of which was important... 

As mentioned earlier, by far the largest number of zinc enzymes are involved in hydrolytic reactions, frequently associated with peptide bond cleavage. Carboxypeptidases and ther-molysins are, respectively, exopeptidases, which remove amino acids from the carboxyl terminus of proteins, and endopeptidases, which cleave peptide bonds in the interior of a polypeptide chain. However, they both have almost identical active sites (Figure 12.4) with two His and one Glu ligands to the Zn2+. It appears that the Glu residue can be bound in a mono- or bi-dentate manner. The two classes of enzymes are expected to follow similar reaction mechanisms. 

The stereochemistry of reactions at zinc atoms has been studied in small molecules (Auf der Heyde and Nassimbeni, 1984) and in proteins (Holmes and Matthews, 1981 Vallee and Auld, 1990a,b). Zinc enzymes include carboxypeptidase A (Quiocho and Lipscomb, 1971 Rees et al., 1983), in which the zinc is coordinated to two histidine nitrogen atoms, two glutamate oxygen atoms, and water (involved in hydrolysis) (Fig. 26). 

Metal ions, especially Zn(II), play an important role in many enzyme-catalyzed reactions involving nucleic acids, such as DNA cleavage by zinc nuclease. Therefore, the binding of Zn(II) to a 19-mer double-stranded oligodeoxyribonucleotide was investigated to understand the role of zinc in DNA cleavage catalyzed by mung bean nuclease [107]. 

This enzyme is a non-specific phosphomonoesterase that shows maximum activity at pH values greater than 8.569 It also catalyzes the transfer of phosphoryl groups. These reactions involve the formation of a phosphoseryl intermediate and the hydrolyzed substrate. The phosphoenzyme may transfer the phosphoryl group to water or to an acceptor molecule to give a new phosphoester (equations 19 and 20, where E—P represents the covalently bound phosphoenzyme and E-P a non-covalent complex, in which phosphate is coordinated to the zinc). The phosphoenzyme may be formed from either direction. 

The N-hydroxy amino acid derivatives are likely to be applicable to other metalloproteases. Thermolysin is inhibited irreversibly at pH 7.2 by ClCH2CO-DL-HOLeu-OCH3 where HOLeu is N-hydroxyleucine (47). The inhibition reaction involves coordination of the hydroxamic acid functional group to the active-site zinc atom of the enzyme. This then places the chloroacetyl group adjacent to Glu-143, an essential catalytic residue of thermolysin (see Figure 9). An ester linkage is formed and the enzyme is inactivated irreversibly. This reagent also inactivated two neutral metalloproteases from B. subtilis, but reacted only very slowly with carboxypeptidase A (t1/2 > 3 d). 

The phenomenological fitting processes yielded 7 = 5.6 kcal mol and w = 10.0 kcal mol h The estimated value of X appears to be in conflict with the value deduced from microscopic computer simulation studies X 80 kcal moH in Ref [64]). Furthermore, the large value of w is hard to rationalize, since the reaction involves a proton transfer between a relatively fixed donor and acceptor (residue 64 and the zinc bound hydroxide). The very small value of X obtained by fitting Eq. (8.20) to experiment is not exclusive to CA III. Similarly, small values were obtained in analysis of other enzymes and are drastically different than the values obtained by actual microscopic computer simulations (note in this respect that X cannot be measured directly). 

Figure 1 shows mechanisms of two enzymes which use a metal ion catalytically. We are going to consider model reactions involving a metal ion and some other possible functional groups. Carboxypeptidase has been described as having a simple set of functional groups required for the activity of the enzyme there is a zinc ion which is coordinated to the... 

Radiometric Assays Traditionally, sulfonated metabolites for many small chemicals have been detected with a radiometric assay that utilizes S -labeled PAPS (Anderson and Weinshilboum, 1980 Foldes and Meek, 1973). The reaction involves incubation of the substrate, cosubstrate, and enzyme in an appropriate buffer. The incubation is terminated by the addition of barium hydroxide, barium acetate, and zinc sulfate, which cause the unreacted PAPS to precipitate out. Thus, the unprecipitated radioactivity is associated with the sulfonated product and can be quantitated with liquid scintillation counting. A variation of this assay has been developed for larger molecules such as flavonoids, where the incubation is terminated by the addition of ethyl acetate under acidic pH conditions and in the presence of an ion-pairing agent, whereby the sulfonated product can then be detected in the organic phase upon liquid-liquid phase separation (Varin et al., 1987). 

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