Metal enzyme binding

A. (The gas phase estimate is about 100 picoseconds for A at 1 atm pressure.) This suggests tliat tire great majority of fast bimolecular processes, e.g., ionic associations, acid-base reactions, metal complexations and ligand-enzyme binding reactions, as well as many slower reactions that are rate limited by a transition state barrier can be conveniently studied with fast transient metliods. 

Many enzymes require metal ions for maximal activity. If the enzyme binds the metal very tightly or requires the metal ion to maintain its stable, native state, it is referred to as a metalloenzyme. Enzymes that bind metal ions more weakly, perhaps only during the catalytic cycle, are referred to as metal activated. One role for metals in metal-activated enzymes and metalloenzymes is to act as electrophilic catalysts, stabilizing the increased electron density or negative charge that can develop during reactions. Among the enzymes that function in this... 

Heavy metals with no known biological function, such as aluminum, arsenic, lead, and mercury, are nonessential metals.4-5 These metals are toxic because they can irreversibly bind to enzymes that require metal cofactors. Toxic metals readily bind to sulfhydryl groups of proteins.6-7 In fact,... 

Moving from single microorganisms to microbial communities, metals create selection pressure for microbes with cell structures that are less sensitive to metals. For example, mutations may occur that alter metal-binding sites of proteins without rendering the enzyme inactive. Another method for preventing metal toxicity is to produce excess amounts of the target so that there is an insufficient amount of metal to bind to all of the cellular molecules.4 35 53... 

An essential aspect to understanding the influence of metal ions on enzyme-catalyzed reactions is the knowledge of how tight different metal ions bind to a wide variety of substrates (particularly nucleotides and other phosphoryl-containingmolecular entities), products, and effectors and that binding phenomena are altered by the experimental conditions (e.g., the effects of pH, temperature, ionic strength, etc.). This necessitates the experimental determination of the stability constant (an association constant) for the metal ion-hgand complex. O Sulhvan and Smithers have reviewed the theory and the various techniques for such determinations and have provided values for many of the more common, biochemically relevant complexes. 

Because metal ions bind to and modify the reactivity and structure of enzymes and substrates, a wide spectrum of techniques has been developed to examine the nature of metal ions which serve as templates, redox-active cofactors, Lewis acids/bases, ion-complexing agents, etc. 

Affinity Labeling of Catalytic ATP Sites. Residues involved in ATP binding are potentially revealed by the use of affinity labels that are based on ATP s structure. Perhaps the most systematically studied of these compounds is 5 -fluorosulfonylbenzoyladenosine (5 -FSBA) (Figure 3a), which has been reported to label at least six kinases (32-A1). In the case of rabbit muscle pyruvate kinase such work has Indicated the presence of a tyrosine residue within the metal nucleotide binding site and an essential cysteine residue located at or near the free metal binding site (32). A similar reagent, 5 -FSBGuanosine, revealed the presence of two cysteine residues at the catalytic site of this same enzyme, both distinct residues from those modified by 5 -FSBA (33,34). With yeast pyruvate kinase both tyrosine and cysteine residues were modified by 5 -FSBA at the catalytic site ( ), and with porcine cAMP-dependent protein kinase a lysine residue was labeled at the active site (36). 

Since then, a considerable amount of structural and mechanistic information has been collected and yeast enolase is probably the best understood sequential enzyme to date. It is a homodimer and requires two Mg + ions per active site for catalytic activity under physiological conditions, although magnesium can be replaced with a variety of divalent metal ions in vitro. During a catalytic turnover, the metal ions bind to the active site in a kinetically ordered, sequential manner with differential binding affinities. The mode of action of yeast enolase is illustrated in Figure 26 and is unusually well understood since several solid-state structures for each intermediate identified with kinetic methods have been determined. 

The binding of small molecules to larger ones is basic to most biological phenomena. Substrates bind to enzymes and hormones bind to receptors. Metal ions bind to ATP, to other small molecules, and to metalloproteins. Hydrogen ions bind to amino acids, peptides, nucleotides, and most macromolecules. In this section we will consider ways of describing mathematically the equilibria involved. 

The 2.9 A resolution structure reveals that the NADH is bound in the enzyme binding domains by an extensive series of hydrogen bonds through the oxygen atoms of the pyrophosphate and the amide group of the nicotinamide. However, no atom in the coenzyme molecule is within bonding distance of the active site zinc atom, the closest being 4.5 A from the metal ion. 

Thus, we have determined the distances between the adenylyl moiety and the two divalent metal ion binding sites on glutamine synthetase by 13C and 3 P NMR, spin-labeled EPR, and fluorescence energy transfer methods. The results obtained from each method are in good agreement. The data show that the adenylyl regulatory site is close to the catalytic site (12-20 A). Additional data on the rotational correlation time of the adenyl derivatives reveal that the adenylyl site is located on the surface of the enzyme. 

This book attempts to describe alternative approaches to ligand reactivity involving normal co-ordination complexes as opposed to organometallic compounds. In part, a justification for this view comes from a study of natural systems. With very few exceptions, organometallic compounds are not involved in biological systems it is equally true that numerous enzymes bind or require metal ions that are essential for their activity. If enzymes can utilise metal ions to perform complex and demanding organic chemical reactions in aqueous, aerobic conditions at ambient temperature and pressure, it would seem to be worthwhile to ask the question whether this is a better approach to catalysis. 

Figure I. Scatchard plots of metal ion binding to PPase as measured by equilibrium dialysis. A Mn2 binding (a) no added Pi, enzyme concentration 7-42/uM (O) (b) in the presence of 50/uM Pi, enzyme concentration 7-36pM ( ) (c) in the presence of 4mM Pi, enzyme concentration 8-116 p M (O). B Co2 binding (a) no added Pi, enzyme concentration 8-96pM (o) (b) in the presence of 2.5mM Pi, enzyme concentration 30-35p M (U) (see Ref. 9).

It has been suggested either that the metal ion binds to co-ordinating groups on the enzyme, e.g. the imidazoles of histidine residues which form part of the active site, or that the metal complex has or can easily adopt a substrate-like structure. 

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