We analysed the roles and distribution of metal ions in enzymatic catalysis using available public databases and our new resource Metal-MACiE (http://www.ebi.ac.uk/thornton-srv/databases/Metal_MACiE/home.html). In Metal-MACiE, a database of metal-based reaction mechanisms, 116 entries covering 21% of the metal-dependent enzymes and 70% of the types of enzyme-catalysed chemical transformations are annotated according to metal function. We used Metal-MACiE to assess the functions performed by metals in biological catalysis and the relative frequencies of different metals in different roles, which can be related to their individual chemical properties and availability in the environment. The overall picture emerging from the overview of Metal-MACiE is that redox-inert metal ions are used in enzymes to stabilize negative charges and to activate substrates by virtue of their Lewis acid properties, whereas redox-active metal ions can be used both as Lewis acids and as redox centres. Magnesium and zinc are by far the most common ions of the first type, while calcium is relatively less used. Magnesium, however, is most often bound to phosphate groups of substrates and interacts with the enzyme only transiently, whereas the other metals are stably bound to the enzyme. The most common metal of the second type is iron, which is prevalent in the catalysis of redox reactions, followed by manganese, cobalt, molybdenum, copper and nickel. The control of the reactivity of redox-active metal ions may involve their association with organic cofactors to form stable units. This occurs sometimes for iron and nickel, and quite often for cobalt and molybdenum.

Metal ions in biological catalysis: from enzyme databases to general principles

Metalloaminopeptidases remove the N-terminal amino acid from proteins or small oligopeptides. This modification or truncation process can occur either co-translationally or posttranslationally after the action of an endoproteinase. The action or inaction of these exopeptidases is instrumental for many normal and pathophysiological processes. The metalloaminopeptidases utilize conserved amino acid residues to generate a scaffold capable of binding one or two metal ions. The metal centers can be described as [M1M2(enzyme)], where M1 and M2 indicate the type of metal ion in site 1 and site 2 for a particular enzyme. Spectroscopic and kinetic analyses indicate that for some of the enzymes two metal ions are required for full activity. In other instances, only a single metal ion is essential for catalysis while the second metal ion modulates activity either positively or negatively. These mononuclear and cocatalytic,1 dinuclear metal centers mediate catalysis by providing sites for substrate binding, by activating the nucleophile of the reaction, and by stabilizing the transition state. Structural analyses of representatives of the different metalloaminopeptidase classes indicate that the site 1 metal ion is closest to the mouth of the active site while the site 2 ion is relatively buried and adjacent to the binding pocket for the P1 side chain (nomenclature of Schechter and Berger2). The enzymes can be categorized based on their substrate specificity or preferences.3 Cleavage of the scissile peptide bond is dependent upon the chemical structure and composition of the amino acid residues that flank the cleavage site (P1*P1′). The broad-range aminopeptidases are typified by the bovine lens leucine aminopeptidase (bLeuAP) and the aminopeptidases from Aeromonas proteolytica (ApAP) and Streptomyces griseus (SgAP). These enzymes are capable of cleaving hydrophobic amino acids from the N-terminus, usually without strict requirements for the penultimate (P1′) residue. In contrast, the “pitabread” enzymes methionine aminopeptidase (MetAP), aminopeptidase P (APP), and prolidase process substrates restricted to particular N-terminal and penultimate residues. This review describes the contributions of many laboratories as they have sought to understand the molecular basis for substrate specificity and catalysis. An effort has been made to give sufficient historical background to appreciate the current state of knowledge. The structure-function relationships for each § Present address: Department of Biochemistry, Wake Forest University School of Medicine, Medical Center Boulevard, WinstonSalem, NC 27157 4581 Chem. Rev. 2002, 102, 4581−4607

Metalloaminopeptidases: common functional themes in disparate structural surroundings.

Fluoride is widely used as an anticaries agent in drinking water and a variety of other vehicles. This use has resulted in major health benefits. However, there are still open questions regarding the mechanisms of anticaries action and the importance of antimicrobial effects in caries reduction. Fluoride acts in multiple ways to affect the metabolism of cariogenic and other bacteria in the mouth. F−/HF can bind directly to many enzymes, for example, heme-containing enzymes or other metalloenzymes, to modulate metabolism. Fluoride is able also to form complexes with metals such as aluminum or beryllium, and the complexes, notably AlF4− and BeF3−·H2O, can mimic phosphate with either positive or negative effects on a variety of enzymes and regulatory phosphatases. The fluoride action that appears to be most important for glycolytic inhibition at low pH in dental plaque bacteria derives from its weak-acid properties (pKa=3.15) and the capacity of HF to act as a transmembrane proton conductor. Since many of the actions of fluoride are related to its weak-acid character, it is reasonable to compare fluoride action to those of organic weak acids, including metabolic acids, food preservatives, non-steroidal anti-inflammatory agents and fatty acids, all of which act to de-energize the cell membrane by discharging ΔpH. Moreover, with the realization that the biofilm state is the common lifestyle for most microorganisms in nature, there is need to consider interactions of fluoride and organic weak acids with biofilm communities. Hopefully, this review will stimulate interest in the antimicrobial effects of fluoride or other weak acids and lead to more effective use of the agents for disease control and other applications.

http://dzumenvis.nic.in/microbes and metals interaction/pdf/fluoride and organic weak acids.pdf

Fluoride and organic weak acids as modulators of microbial physiology.

Structure and function of the methionine aminopeptidases.

Mode of action of bi- and trinuclear zinc hydrolases and their synthetic analogues.

The aminopeptidase from Aeromonas proteolytica (AAP) is uncompetitively inhibited by fluoride ion at pH 8.0 with an inhibition constant (Ki) of 30 mM. Thus, fluoride inactivates AAP only after subs...

https://epublications.marquette.edu/cgi/viewcontent.cgi?article=1301&context=chem_fac

Mechanistic Studies on the Aminopeptidase from Aeromonas proteolytica : A Two-Metal Ion Mechanism for Peptide Hydrolysis

The nature of the interaction of the transition-state analogue inhibitor L-leucinephosphonic acid (LPA) with the leucine aminopeptidase from Aeromonas proteolytica (AAP) was investigated. LPA was shown to be a competitive inhibitor at pH 8.0 with a K(i) of 6.6 microM. Electronic absorption spectra, recorded at pH 7.5 of [CoCo(AAP)], [CoZn(AAP)], and [ZnCo(AAP)] upon addition of LPA suggest that LPA interacts with both metal ions in the dinuclear active site. EPR studies on the Co(II)-substituted forms of AAP revealed that the environments of the Co(II) ions in both [CoZn(AAP)] and [ZnCo(AAP)] become highly asymmetric and constrained upon the addition of LPA and clearly indicate that LPA interacts with both metal ions. The X-ray crystal structure of AAP complexed with LPA was determined at 2.1 A resolution. The X-ray crystallographic data indicate that LPA interacts with both metal centers in the dinuclear active site of AAP and a single oxygen atom bridge is absent. Thus, LPA binds to the dinuclear active site of AAP as an eta-1,2-mu-phosphonate with one ligand to the second metal ion provided by the N-terminal amine. A structural comparison of the binding of phosphonate-containing transition-state analogues to the mono- and bimetallic peptidases provides insight into the requirement for the second metal ion in bridged bimetallic peptidases. On the basis of the results obtained from the spectroscopic and X-ray crystallographic data presented herein along with previously reported mechanistic data for AAP, a new catalytic mechanism for the hydrolysis reaction catalyzed by AAP is proposed.

/pdf/inhibition-of-the-aminopeptidase-from-aeromonas-proteolytica-4qyt5873e6.pdf

Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis.

Seven aliphatic and two aromatic alcohols were tested as reporters of the substrate selectivity of the aminopeptidase from Aeromonas proteolytica (AAP). This series of alcohols was chosen to systematically probe the effect of carbon chain length, steric bulk, and inhibitor shape on the inhibition of AAP. Initially, however, the question of whether AAP is denatured in the presence of aliphatic alcohols was addressed. On the basis of circular dichroism (CD), electronic absorption, and fluorescence spectra, the secondary structure of AAP, with and without added aliphatic alcohols, was unchanged. These data clearly indicate that AAP is not denatured in aliphatic alcohols, even up to concentrations of 20% (v/v). All of the alcohols studied were competitive inhibitors of AAP with K(i) values between 860 and 0.98 mM. The clear trend in the data was that as the carbon chain length increases from one to four, the K(i) values increase. Branching of the carbon chains also increases the K(i) values, but large bulky groups, such as that found in tert-butyl alcohol, do not inhibit AAP as well as leucine analogues, such as 3-methyl-1-butanol. The competitive nature of the inhibition indicates that the substrate and each alcohol studied are mutually exclusive due to binding at the same site on the enzyme. On the basis of EPR and electronic absorption data for Co(II)-substituted AAP, none of the alcohols studied binds to the dinuclear metallo-active site of AAP. Thus, reaction of the inhibitory alcohols with the catalytic metal ions cannot constitute the mechanism of inhibition. Combination of these data suggests that each of these inhibitors bind only to the hydrophobic pocket of AAP and, consequently, block the binding of substrate. Thus, the first step in peptide hydrolysis is the recognition of the N-terminal amino acid side chain by the hydrophobic pocket adjacent to the dinuclear active site of AAP.

/pdf/inhibition-of-the-aminopeptidase-from-aeromonas-proteolytica-3t2xv4l3cm.pdf

Tanya Edwards

Papers

Mechanistic Studies on the Aminopeptidase from Aeromonas proteolytica : A Two-Metal Ion Mechanism for Peptide Hydrolysis

Inhibition of the aminopeptidase from Aeromonas proteolytica by L-leucinephosphonic acid. Spectroscopic and crystallographic characterization of the transition state of peptide hydrolysis.

Inhibition of the aminopeptidase from Aeromonas proteolytica by aliphatic alcohols. Characterization of the hydrophobic substrate recognition site.