Mechanisms of peptide hydrolysis by aspartyl and metalloproteases.

Peptide hydrolysis has been involved in a wide range of biological, biotechnological, and industrial applications. In this perspective, the mechanisms of three distinct peptide bond cleaving enzymes, beta secretase (BACE1), insulin degrading enzyme (IDE), and bovine lens leucine aminopeptidase (BILAP), have been discussed. BACE1 is a catalytic Asp dyad [Asp, Asp-] containing aspartyl protease, while IDE and BILAP are mononuclear [Zn(His, His, Glu)] and binuclear [Zn1(Asp, Glu, Asp)-Zn2(Lys, Glu, Asp, Asp)] core possessing metallopeptidases, respectively. Specifically, enzyme-substrate interactions and the roles of metal ion(s), the ligand environment, second coordination shell residues, and the protein environment in the functioning of these enzymes have been elucidated. This information will be useful to design small inhibitors, activators, and synthetic analogues of these enzymes for biomedical, biotechnological, and industrial applications.

Coupled Dynamics and Entropic Contribution to the Allosteric Mechanism of Pin1.

Allosteric communication in proteins regulates a plethora of downstream processes in subcellular signaling pathways. Describing the effects of cooperative ligand binding on the atomic level is a key to understanding many regulatory processes involving biomolecules. Here, we use microsecond-long molecular dynamics simulations to investigate the allosteric mechanism of Pin1, a potential therapeutic target and a phosphorylated-Ser/Thr dependent peptidyl-prolyl cis-trans isomerase that regulates several subcellular processes and has been implicated in many diseases, including cancer and Alzheimer's. Experimental studies suggest that the catalytic domain and the noncatalytic WW domain are allosterically coupled; however, an atomic level description of the dynamics associated with the interdomain communication is lacking. We show that binding of the substrate to the WW domain is directly coupled to the dynamics of the catalytic domain, causing rearrangement of the residue-residue contact dynamics from the WW domain to the catalytic domain. The binding affinity of the substrate in the catalytic domain is also enhanced upon binding of the substrate to the WW domain. Modulation of the dynamics of the catalytic domain upon binding of the substrate to the WW domain leads to prepayment of the entropic cost of binding the substrate to the catalytic domain. This study shows that Ile 28 at the interfacial region between the catalytic and WW domains is certainly one of the residues responsible for bridging the communication between the two domains. The results complement previous experiments and provide valuable atomistic insights into the role of dynamics and possible entropic contribution to the allosteric mechanism of proteins.

An Iron Reservoir to the Catalytic Metal: THE RUBREDOXIN IRON IN AN EXTRADIOL DIOXYGENASE.

The rubredoxin motif is present in over 74,000 protein sequences and 2,000 structures, but few have known functions. A secondary, non-catalytic, rubredoxin-like iron site is conserved in 3-hydroxyanthranilate 3,4-dioxygenase (HAO), from single cellular sources but not multicellular sources. Through the population of the two metal binding sites with various metals in bacterial HAO, the structural and functional relationship of the rubredoxin-like site was investigated using kinetic, spectroscopic, crystallographic, and computational approaches. It is shown that the first metal presented preferentially binds to the catalytic site rather than the rubredoxin-like site, which selectively binds iron when the catalytic site is occupied. Furthermore, an iron ion bound to the rubredoxin-like site is readily delivered to an empty catalytic site of metal-free HAO via an intermolecular transfer mechanism. Through the use of metal analysis and catalytic activity measurements, we show that a downstream metabolic intermediate can selectively remove the catalytic iron. As the prokaryotic HAO is often crucial for cell survival, there is a need for ensuring its activity. These results suggest that the rubredoxin-like site is a possible auxiliary iron source to the catalytic center when it is lost during catalysis in a pathway with metabolic intermediates of metal-chelating properties. A spare tire concept is proposed based on this biochemical study, and this concept opens up a potentially new functional paradigm for iron-sulfur centers in iron-dependent enzymes as transient iron binding and shuttling sites to ensure full metal loading of the catalytic site.

Theoretical insights into the functioning of metallopeptidases and their synthetic analogues.

CONSPECTUS: The selective hydrolysis of a peptide or amide bond (-(O═)C-NH-) by a synthetic metallopeptidase is required in a wide range of biological, biotechnological, and industrial applications. In nature, highly specialized enzymes known as proteases and peptidases are used to accomplish this daunting task. Currently, many peptide bond cleaving enzymes and synthetic reagents have been utilized to achieve efficient peptide hydrolysis. However, they possess some serious limitations. To overcome these inadequacies, a variety of metal complexes have been developed that mimic the activities of natural enzymes (metallopeptidases). However, in comparison to metallopeptidases, the hydrolytic reactions facilitated by their existing synthetic analogues are considerably slower and occur with lower catalytic turnover. This could be due to the following reasons: (1) they lack chemical properties of amino acid residues found within enzyme active sites; (2) they contain a higher metal coordination number compared with naturally occurring enzymes; and (3) they do not have access to second coordination shell residues that provide substantial rate enhancements in enzymes. Additionally, the critical structural and mechanistic information required for the development of the next generation of synthetic metallopeptidases cannot be readily obtained through existing experimental techniques. This is because most experimental techniques cannot follow the individual chemical steps in the catalytic cycle due to the fast rate of enzymes. They are also limited by the fact that the diamagnetic d(10) Zn(II) center is silent to electronic, electron spin resonance, and (67)Zn NMR spectroscopies. Therefore, we have employed molecular dynamics (MD), quantum mechanics (QM), and hybrid quantum mechanics/molecular mechanics (QM/MM) techniques to derive this information. In particular, the role of the metal ions, ligands, and microenvironment in the functioning of mono- and binuclear metal center containing enzymes such as insulin degrading enzyme (IDE) and bovine lens leucine aminopeptidase (BILAP), respectively, and their synthetic analogues have been investigated. Our results suggested that in the functioning of IDE, the chemical nature of the peptide bond played a role in the energetics of the reaction and the peptide bond cleavage occurred in the rate-limiting step of the mechanism. In the cocatalytic mechanism used by BILAP, one metal center polarized the scissile peptide bond through the formation of a bond between the metal and the carbonyl group of the substrate, while the second metal center delivered the hydroxyl nucleophile. The Zn(N3) [Zn(His, His, His)] core of matrix metalloproteinase was better than the Zn(N2O) [Zn(His, His, Glu)] core of IDE for peptide hydrolysis. Due to the synergistic interaction between the two metal centers, the binuclear metal center containing Pd2(µ-OH)([18]aneN6)](4+) complex was found to be ∼100 times faster than the mononuclear [Pd(H2O)4](2+) complex. A successful small-molecule synthetic analogue of a mononuclear metallopeptidase must contain a metal with a strong Lewis acidity capable of reducing the pKa of its water ligand to less than 7. Ideally, the metal center should include three ligands with low basicity. The steric effects or strain exerted by the microenvironment could be used to weaken the metal-ligand interactions and increase the activity of the metallopeptidase.

Cysteine-mediated dynamic hydrogen-bonding network in the active site of Pin1.

Enzymes catalyze a plethora of chemical reactions that are tightly regulated and intricately coupled in biology. Catalysis of phosphorylation-dependent cis-trans isomerization of peptidyl-prolyl bonds, which act as conformational switches in regulating many post-phosphorylation processes, is considered to be one of the most critical. Pin1 is a cis-trans isomerase of peptidyl-prolyl(ω-) bonds of phosphorylated-Ser/Thr-Pro motifs and has been implicated in many diseases. Structural and experimental studies are still unable to resolve the mechanistic role and protonation states of two adjacent histidines (His59 and His157) and a cysteine (Cys113) in the active site of Pin1. Here, we show that the protonation state of Cys113 mediates a dynamic hydrogen-bonding network in the active site of Pin1, involving the two adjacent histidines and several other residues that are highly conserved and necessary for catalysis. We have used detailed free energy calculations and molecular dynamics simulations, complementing previous experiments, to resolve the ambiguities in the orientations of the histidines and protonation states of these key active site residues, details that are critical for fully understanding the mechanism of Pin1 and necessary for developing potent inhibitors. Importantly, Cys113 is shown to alternate between the unprotonated and neutral states, unprotonated in free Pin1 and neutral in substrate-bound Pin1. Our results are consistent with experiments and provide an explanation for the chemical reactivity of free Pin1 that is suggested to be necessary for the regulation of the enzyme.

Elucidating the catalytic mechanism of ß-secretase (BACE1): a quantum mechanics/molecular mechanics (QM/MM) approach.

In this quantum mechanics/molecular mechanics (QM/MM) study, the mechanisms of the hydrolytic cleavage of the Met2-Asp3 and Leu2-Asp3 peptide bonds of the amyloid precursor protein (WT-substrate) and its Swedish mutant (SW) respectively catalyzed by ß-secretase (BACE1) have been investigated by explicitly including the electrostatic and steric effects of the protein environment in the calculations. BACE1 catalyzes the rate-determining step in the generation of Alzheimer amyloid beta peptides and is widely acknowledged as a promising therapeutic target. The general acid-base mechanism followed by the enzyme proceeds through the following two steps: (1) formation of the gem-diol intermediate and (2) cleavage of the peptide bond. The formation of the gem-diol intermediate occurs with the barriers of 19.6 and 16.1 kcal/mol for the WT- and SW-substrate respectively. The QM/MM energetics predict that with the barriers of 21.9 and 17.2 kcal/mol for the WT- and SW-substrate respectively the cleavage of the peptide bond occurs in the rate-determining step. The computed barriers are in excellent agreement with the measured barrier of ∼18.0 kcal/mol for the SW-substrate and in line with the experimental observation that the cleavage of this substrate is sixty times more efficient than the WT-substrate.

Mechanism of peptide hydrolysis by co-catalytic metal centers containing leucine aminopeptidase enzyme: a DFT approach.

In this density functional theory study, reaction mechanisms of a co-catalytic binuclear metal center (Zn1-Zn2) containing enzyme leucine aminopeptidase for two different metal bridging nucleophiles (H(2)O and -OH) have been investigated. In addition, the effects of the substrate (L-leucine-p-nitroanilide → L-leucyl-p-anisidine) and metal (Zn1 → Mg and Zn2 → Co, i.e., Mg1-Zn2 and Mg1-Co2 variants) substitutions on the energetics of the mechanism have been investigated. The general acid/base mechanism utilizing a bicarbonate ion followed by this enzyme is divided into two steps: (1) the formation of the gem-diolate intermediate, and (2) the cleavage of the peptide bond. With the computed barrier of 17.8 kcal/mol, the mechanism utilizing a hydroxyl nucleophile was found to be in excellent agreement with the experimentally measured barrier of 18.7 kcal/mol. The rate-limiting step for reaction with L-leucine-p-nitroanilide is the cleavage of the peptide bond with a barrier of 17.8 kcal/mol. However, for L-leucyl-p-anisidine all steps of the mechanism were found to occur with similar barriers (18.0-19.0 kcal/mol). For the metallovariants, cleavage of the peptide bond occurs in the rate-limiting step with barriers of 17.8, 18.0, and 24.2 kcal/mol for the Zn1-Zn2, Mg1-Zn2, and Mg1-Co2 enzymes, respectively. The nature of the metal ion was found to affect only the creation of the gem-diolate intermediate, and after that all three enzymes follow essentially the same energetics. The results reported in this study have elucidated specific roles of both metal centers, the nucleophile, indirect ligands, and substrates in the catalytic functioning of this important class of binuclear metallopeptidases.

Which one among aspartyl protease, metallopeptidase, and artificial metallopeptidase is the most efficient catalyst in peptide hydrolysis?

In this comparative DFT study, the hydrolysis of a peptide bond (Phe1-Phe2) by the following three types of catalysts has been studied: (1) beta-secretase (BACE2), (2) matrix metalloproteinase (MMP) and insulin degrading enzyme (IDE), and (3) [Pd(H(2)O)(4)](2+) (I(MPC)) and [Pd(2)(mu-OH)([18]aneN(6))](3+) (I(DPC)). The computed energetics predict that among these catalysts, the Zn(2+) metal center containing MMP is the most efficient in catalyzing this reaction. The two active site aspartate residues containing BACE2 catalyze this reaction with 5.0 kcal/mol higher barrier than MMP. The substitution of a His ligand with Glu in the metal center of MMP generates the active site of IDE that catalyzes the reaction with a 6.9 kcal/mol higher barrier than MMP. Both artificial peptidases I(MPC) and I(DPC) catalyze this reaction with significantly high barriers of 35.4 and 31.0 kcal/mol, respectively. The computed energetics of all the catalysts are in line with the available experimental and theoretical data.

Insights into the mechanism of methionine oxidation catalyzed by metal (Cu(2+), Zn(2+), and Fe(3+)) - amyloid beta (Abeta) peptide complexes: A computational study.

In this DFT study, a mechanism of the oxidation of methionine (Met) amino acid residue catalyzed by the metal (Cu(2+), Zn(2+), and Fe(3+)) bound amyloid beta (Abeta) peptide has been proposed. Based on experimental information, two different mechanisms: (1) stepwise and (2) concerted mechanisms for this important process have been investigated. The B3LYP calculations suggest that in the stepwise mechanism, the two separate pathways leading to the same sulfoxide product [Met(O)] go through prohibitively high barriers of 27.3 and 35.1 kcal/mol, therefore it is ruled out. In the concerted mechanism, the Cu(2+)-Abeta complex has been found to be the most efficient catalyst with the computed barrier of 14.3 kcal/mol. The substitutions of Cu(2+) by Zn(2+) and Fe(3+) increase barriers to 19.6 and 16.9 kcal/mol, respectively and make the reaction thermodynamically less favorable. It was also found that, in comparison with the cysteine (Cys) residue, Met is more susceptible toward oxidation. Its substitution with Cys slightly increased the barrier to 15.8 kcal/mol for the Cu(2+)-Abeta complex.