Metalloproteases

Background

Thermolysin (TLN; EC 3.4.24.27, Figure 1) is a 34.6 kDa Zn²⁺-endopeptidase secreted by the bacterium Bacillus thermoproteolyticus.^8,9 TLN and TLN-like proteins are used by bacteria to break down exogenous proteins for nutrition and as virulence factors aiding in host colonization and tissue degradation.^10,11 TLN is active in the hydrolysis of internal peptide bonds on the N-terminal side of large hydrophobic amino acids, between leucine, isoleucine, or phenylalanine. Thermolysin was the first metalloproteases to be completely sequenced.¹² Commercially, TLN is used as a nonspecific protease (within its cleavage site specificity) in peptide sequencing and is used in the production of the artificial sweetener aspartame.¹³

Figure 1. General structure of thermolysin (PDBID 3DNZ). Alpha-helices in blue, beta-sheets in red, Zn²⁺ ion in yellow, active site side chains in magenta, Ca²⁺ ions in green.⁸

Structure

The overall structure of the protein consists of 316 amino acid residues organized into two domains separated by the active site.¹² The N-terminal domain is predominately composed of β-sheets, while the C-terminal domain is primarily composed of α-helices. The two large domains are separated by a central α-helix. One Zn²⁺ is bound via three residues in the active site within a conserved binding motif of HEXXH (His 142, His 146, Glu 166) located on the C-terminal side (Figure 2).^14,15 The tetrahedral binding of Zn²⁺ is rounded out by a nucleophilic water molecule. This water molecule is capable of alternating between two distinct association positions, W1 and W2, which are in turn stabilized by His 231 and Glu 143, respectively.¹⁶ Four Ca2+ ions are associated with the enzyme that aide in thermostability.¹²

Figure 2. Active site of thermolysin.

Catalytic Residue Importance

Zinc: The bound Zn²⁺ is responsible for catalyzing peptide hydrolysis and stabilizing the various intermediates of the reaction. Although normally bound in a tetrahedral structure, during catalysis Zn²⁺ assumes a pentacoordinate geometry between the original three residues (His142, His146, and Glu166), the oxygen of the nucleophilic water, and the carbonyl oxygen of the substrate.^7,14,17 The formation of a gem-diolate intermediate is stabilized by Zn²⁺.^14,17 Removal of Zn²⁺ yields an inactive enzyme. Exogenous addition of other divalent transition metals, specifically Zn²⁺_, Co²⁺, Fe²⁺, and Mn²⁺, results in the regaining of 100%, 200%, 60%, and 10% enzymatic activity.³ Zn²⁺ is also responsible for the polarization of the carbonyl bond of the substrate and the enhancement of nucleophilicity of the catalytic water molecule.

Glu143: Glu143 is responsible for the polarization of the catalytic water molecule leading to an enhancement of nucleophilicity. Additionally, Glu143 abstracts a proton from the water molecule and transfers it to the amide leaving group. In site-directed mutagenesis experiments involving the neutral protease of B. stearothermophilus, a protease with an amino acid sequence identical to that of thermolysin, Glu143Asp and Glu143Gln substitutions resulted in no catalytic activity of the enzyme.^12,18 The mechanism proposed by Mock and colleagues suggests a diminished catalytic role of Glu143, which is instead used solely for charge stabilization with no association with the nucleophilic water molecule.^19,20 Mutagenesis studies show that Glu143Asp substitutions result in inactive enzymes, despite the same side chain charge that would provide electrostatic stability.¹⁸

His231: His231 is responsible for substrate stabilization during hydrolysis. The carbonyl oxygen of the peptide is hydrogen bonded to N₁ of His231 in the intermediate step of hydrolysis. Mutagenesis experiments involving His231Phe and His231Ala show 430- and 500-fold reductions in catalytic activity with no significant change in K_m.²¹ These findings led to a proposed TLN-mechanism with an emphasis on the role of His231 as a general base, but this mechanism is less favored due to the residual activities of His231-substituted mutants compared to inactive mutants generated by Glu143 mutagenesis studies.^18,19,21

Tyr157: The importance of Tyr157 has been debated in several mechanisms.^18,22 Site-directed mutagenesis studies show an 80% drop in catalytic activity of Tyr157Trp mutants, and it has been suggested that the hydroxyl-H of Ty157 stabilizes the carboxylate-O of the peptide substrate during hydrolysis.^17,23 Energetics modeling suggests a primary role for Tyr157 in transition state stabilization and substrate binding with an increase in the activation barrier energy of 2.7 kcal/mol and a decrease of binding affinity of 0.5 kcal/mol.²⁴

Asp226: Asp226 has been suggested to stabilize the catalytic His231 through H-bonding of the Asp226 carboxylate to N₃ of His231.^17,22 Mutagenesis of Asp226Ala shows a relatively small decrease in catalytic activity of 40-fold and energetics modeling suggest an increase in overall ΔG‡ 2.2 kcal/mol.^23,24

Mechanism

Scheme 1. Mechanism of peptide hydrolysis by thermolysin.²⁴

Several mechanisms for TLN mediated peptide cleavage have been proposed with varying emphasis on the importance of the roles of the catalytic residues, Glu143 and His231, as well as several catalysis-associated residues, Tyr157 and Asp226. The generally accepted mechanism for TLN-mediated hydrolysis proceeds via the two-step process depicted in Scheme 1.²⁴ Briefly:

1. The active site houses the catalytic Zn²⁺ ion bound to three residues and a catalytic water (W)
2. An incoming peptide substrate displaces the hydrogens of W₁ towards Glu143, while the oxygen of W (O_W) remains associated with Zn²⁺; Zn²⁺ associates with the carbonyl oxygen (O_P) of the substrate forming the enzyme-substrate complex (ES)
3. Polarizing actions on Zn²⁺ by Glu143 allow nucleophilic attack of carbonyl carbon (C_P) by O_W forming Transition State 1 (TS1); stabilized by Asp226 at N₃, His231 N₁ (originally protonated) stabilizes the substrate via hydrogen bonding at the carbonyl oxygen
4. The intermediate (INT) gem-diolate is formed as Glu143 breaks the H_W1-O_W bond and the amide of the substrate (N_P) hydrogen bonds H_W2
5. The N_P-C_P bond of the substrate begins to break as the C_P-O_P bond regains double bond character and the tetrahedral C_P gem-diolate intermediate is pushed to Transition State 2 (TS2)
6. The N_P-C_P is fully broken and the Glu143-bound H_W2 bond begins to break as the new N-terminal hydrolyzed peptide is released (PROD)
8. The active site is hydrated and is free for new peptide docking

The catalytic activity of TLN is dependent on both temperature and pH.²⁵ The pK_as of TLN are 5.0 and 8.25, and maximum catalytic activity has been measured at pH 7.2.^25,26 The thermostability of TLN conferred by the four Ca²⁺ ions allows an increase in catalytic activity at temperatures approaching 40°C, with no significant loss in activity or conformation alteration until temperatures exceed 70°C.^25,27

Inhibition and Inhibitors

Chelating agents such as EDTA have been shown to completely inactivate TLN and other metalloproteases by removal of the metal ion.³ In addition to direct removal of the catalytic metal ion, substrate and transition state analogs have been synthesized that greatly decrease the catalytic activity of TLN and TLN-like proteins. Transition state analogs that resemble the mechanistic transition states of the normal hydrolysis reaction catalyzed by TLN have been both isolated and synthesize. Phosphoramidates, a group of phosphoryl group containing amino acid or peptide compounds have been shown to mimic the tetrahedral intermediate of the TLN mechanism.^28,29 Overall inhibition has been shown to vary from five to 1000-fold decrease in catalytic activity through use of transition state analogs.^28,30 Holmquist and Vallee combined substrate analog inhibition with anionic ligands that tightly bind active site metals such as R-S^- and R-P-O^-.³⁰ By combining two approaches, catalytic activity of TLN was decreased 10,000-fold. This compared to ~800-fold decrease when transition state analogs were combined with metal-binding ligands, suggesting a stronger inhibition effect of the substrate analog.³⁰

Background

Carboxypeptidase A (carboxypolypeptidase; CPA; EC 3.4.17.1) is a 35 kD metalloenzyme within the zinc hydrolase family and as such contains a Zn2+ ion cofactor located within its active site.^31,32 Originally isolated from bovine pancreas tissue in 1929, CPA is an exopeptidase that catalyzes the hydrolysis of C-terminal esters and peptides with large hydrophobic side chains.^31,32 Biologically, CPA facilitates the breakdown of proteins during metabolism, while proposed commercial applications include its use the hydrolysis of cheese whey protein and the production of phenylalanine-free protein hydrolysates for use by individuals with phenylketonuria.^33,34

Figure 3: General structure of carboxypeptidase A (PDBID 1YME). Alpha-helices in blue, beta-sheets in red, Zn²⁺ ion in yellow, active site side chains in cyan.

Figure 4: Carboxypeptidase A active site.³²

Structure

The structure of CPA was first determined in 1967 using x-ray diffraction, making it one earliest proteins to have been characterized using the technique (Figure 3).³⁵ CPA consists of a single chain containing 307 amino acids and a single Zn²⁺ ion in its active site.³⁵ Zn²⁺ is stabilized within the active site through interactions with His69, Glu72, and His196 and is additionally bound by a catalytic water molecule that has been shown to interact with Glu270 (Figure 4).³⁶ Mutagenesis studies have indicated that the additional residues Arg127, Tyr248, Arg71, Asn144, and Arg145 form an outer shell, which includes Glu270, around the active site and contribute to the catalytic function of CPA through the stabilization of substrate molecules.³⁶ In both proposed mechanisms for the catalytic activity of CPA, Glu270 plays an important role, either acting as a general base-general acid or as a nucleophile.³⁶

Mechanisms

Two mechanisms have been suggested for CPA catalyzed hydrolysis—the promoted-water pathway, also known as the general base-general acid pathway, and the nucleophilic, or anhydride, pathway—with experimental evidence existing for both mechanisms.³⁶

Inhibitors and Inhibition

Ultraviolet-visible radiation (400 W, λ=250-750 nm) has been shown to cause uncompetitive inhibition of CPA with the decrease in enzymatic activity indirectly proportional to the irradiation time, with total enzymatic inactivation after 20 minutes of exposure. Additionally, exposure times of greater than 24 minutes are suspected to adversely affect the structure of CPA, resulting in the formation of protein aggregates.³⁷

Active site-directed inhibitors of CPA, which are characterized by the presence of a terminal carboxylate, a hydrophobic side chain, and a zinc-binding group, have been identified, among which include the enantiomers of 2-benzyl-5-hydroxy-4-oxopentanoic acid. (L)-2-benzyl-5-hydroxy-4-oxopentanoic acid interacts through its carboxylate by forming hydrogen bonds with Arg145, Arg127, and Tyr248 and through its terminal hydroxyl group by forming a hydrogen bond with Ser197.³⁸

Transition state analog inhibitors of CPA have also been found, which when bound with the enzyme active site create a pseudo-transition state complex. Both (R)-2-benzyl-3-nitropropanoic acid and (R)-2-benzyl-5-nitro-4-oxopentanoic acid are capable of inhibiting CPA by forming complexes with their respective nitro groups, Glu270, Arg127, and Zn²⁺.^39,40