Triad of polar residues implicated in pH specificity of acidic mammalian chitinase

Acidic mammalian chitinase (AMCase) is a mammalian chitinase that has been implicated in allergic asthma. One of only two active mammalian chinases, AMCase, is distinguished from other chitinases by several unique features. Here, we present the novel structure of the AMCase catalytic domain, both in the apo form and in complex with the inhibitor methylallosamidin, determined to high resolution by X‐ray crystallography. These results provide a structural basis for understanding some of the unique characteristics of this enzyme, including the low pH optimum and the preference for the β‐anomer of the substrate. A triad of polar residues in the second‐shell is found to modulate the highly conserved chitinase active site. As a novel target for asthma therapy, structural details of AMCase activity will help guide the future design of specific and potent AMCase inhibitors.     

Evolutionary and biochemical differences between human and monkey acidic mammalian chitinases

Acidic mammalian chitinase (AMCase), an enzyme implicated in the pathology of asthma, is capable of chitin cleavage at a low pH optimum. The corresponding gene (CHIA) can be found in genome databases of a variety of mammals, but the enzyme properties of only the human and mouse proteins were extensively studied. We wanted to compare enzymes of closely related species, such as humans and macaques. In our attempt to study macaque AMCase, we searched for CHIA-like genes in human and macaque genomes. We found that both genomes contain several additional CHIA-like sequences. In humans, CHIA-L1 (hCHIA-L1) is an apparent pseudogene and has the highest homology to CHIA. To determine which of the two genes is functional in monkeys, we assessed their tissue expression levels. In our experiments, CHIA-L1 expression was not detected in human stomach tissue, while CHIA was expressed at high levels. However, in the cynomolgus macaque stomach tissue, the expression pattern of these two genes was reversed: CHIA-L1 was expressed at high levels and CHIA was undetectable. We hypothesized that in macaques CHIA-L1 (mCHIA-L1), and not CHIA, is a gene encoding an acidic chitinase, and cloned it, using the sequence of human CHIA-L1 as a guide for the primer design. We named the new enzyme MACase (Macaca Acidic Chitinase) to emphasize its differences from AMCase. MACase shares a similar tissue expression pattern and pH optimum with human AMCase, but is 50 times more active in our enzymatic activity assay. DNA sequence of the mCHIA-L1 has higher percentage identity to the human pseudogene hCHIA-L1 (91.7%) than to hCHIA (84%). Our results suggest alternate evolutionary paths for human and monkey acidic chitinases.     

Kinetic characterization of recombinant human acidic mammalian chitinase

Human acidic mammalian chitinase (AMCase), a member of the family 18 glycosyl hydrolases, is one of the important proteins involved in Th2-mediated inflammation and has been implicated in asthma and allergic diseases. Inhibition of AMCase results in decreased airway inflammation and airway hyper-responsiveness in a mouse asthma model, suggesting that the AMCase activity is a part of the mechanism of Th2 cytokine-driven inflammatory response in asthma. In this paper, we report the first detailed kinetic characterization of recombinant human AMCase. In contrast with mouse AMCase that has been reported to have a major pH optimum at 2 and a secondary pH optimum around 3-6, human AMCase has only one pH optimum for k(cat)/K(m) between pH 4 and 5. Steady state kinetics shows that human AMCase has "low" intrinsic transglycosidase activity, which leads to the observation of apparent substrate inhibition. This slow transglycosylation may provide a mechanism in vivo for feedback regulation of the chitinase activity of human AMCase. HPLC characterization of cleavage of chitooligosaccharides (4-6-mers) suggests that human AMCase prefers the beta anomer of chitooligosaccharides as substrate. Human AMCase also appears to cleave chitooligosaccharides from the nonreducing end primarily by disaccharide units. Ionic strength modulates the enzymatic activity and substrate cleavage pattern of human AMCase against fluorogenic substrates, chitobiose-4-methylumbelliferyl and chitotriose-4-methylumbelliferyl, and enhances activity against chitooligosaccharides. The physiological implications of these results are discussed.     

A rationalization of the acidic pH dependence for stromelysin-1 (Matrix metalloproteinase-3) catalysis and inhibition

The pH dependence of matrix metalloproteinase (MMP) catalysis is described by a broad bell-shaped curve, indicating the involvement of two unspecified ionizable groups in proteolysis. Stromelysin-1 has a third pK(a) near 6, resulting in a uniquely sharp acidic catalytic optimum, which has recently been attributed to His(224). This suggests the presence of a critical, but unidentified, S1' substructure. Integrating biochemical characterizations of inhibitor-enzyme interactions with active site topography from corresponding crystal structures, we isolated contributions to the pH dependence of catalysis and inhibition of active site residues Glu(202) and His(224). The acidic pK(a) 5.6 is attributed to the Glu(202).zinc.H(2)O complex, consistent with a role for the invariant active site Glu as a general base in MMP catalysis. The His(224)-dependent substructure is identified as a tripeptide (Pro(221)-Leu(222)-Tyr(223)) that forms the substrate cleft lower wall. Substrate binding induces a beta-conformation in this sequence, which extends and anchors the larger beta-sheet of the enzyme. substrate complex and appears to be essential for productive substrate binding. Because the PXY tripeptide is strictly conserved among MMPs, this "beta-anchor" may represent a common motif required for macromolecular substrate hydrolysis. The striking acidic profile of stromelysin-1 defined by the combined ionization of Glu(202) and His(224) allows the design of highly selective inhibitors.     

Identification of a novel acidic mammalian chitinase distinct from chitotriosidase

Chitinases are ubiquitous chitin-fragmenting hydrolases. Recently we discovered the first human chitinase, named chitotriosidase, that is specifically expressed by phagocytes. We here report the identification, purification, and subsequent cloning of a second mammalian chitinase. This enzyme is characterized by an acidic isoelectric point and therefore named acidic mammalian chitinase (AMCase). In rodents and man the enzyme is relatively abundant in the gastrointestinal tract and is found to a lesser extent in the lung. Like chitotriosidase, AMCase is synthesized as a 50-kDa protein containing a 39-kDa N-terminal catalytic domain, a hinge region, and a C-terminal chitin-binding domain. In contrast to chitotriosidase, the enzyme is extremely acid stable and shows a distinct second pH optimum around pH 2. AMCase is capable of cleaving artificial chitin-like substrates as well as crab shell chitin and chitin as present in the fungal cell wall. Our study has revealed the existence of a chitinolytic enzyme in the gastrointestinal tract and lung that may play a role in digestion and/or defense.     

Activation of lactoperoxidase by heme‐linked protonation and heme‐independent iodide binding

Lactoperoxidase (LPO), a mammalian secretory heme peroxidase, catalyzes the oxidation of thiocyanate by hydrogen peroxide to produce hypothiocyanate, an antibacterial agent. Although LPO is known to be activated at acidic pH and in the presence of iodide, the structural basis of the activation is not well understood. We have examined the effects of pH and iodide concentration on the catalytic activity and the structure of LPO. Electrochemical and colorimetric assays have shown that the catalytic activity is maximized at pH 4.5. The heme Soret absorption band exhibits a small red‐shift at pH 5.0 upon acidification, which is ascribable to a structural transition from a neutral to an acidic form. Resonance Raman spectra suggest that the heme porphyrin core is slightly contracted and the Fe‐His bond is strengthened in the acidic form compared to the neutral form. The structural change of LPO upon activation at acidic pH is similar to that observed for myeloperoxidase, another mammalian heme peroxidase, upon activation at neutral pH. Binding of iodide enhances the catalytic activity of LPO without affecting either the optimum pH of activity or the heme structure, implying that the iodide binding occurs at a protein site away from the heme‐linked protonation site. © 2009 Wiley Periodicals, Inc. Biopolymers 93: 113–120, 2010. This article was originally published online as an accepted preprint. The “Published Online” date corresponds to the preprint version. You can request a copy of the preprint by emailing the Biopolymers editorial office at biopolymers@wiley.com     

Effects of the residue adjacent to the reactive serine on the substrate interactions ofDrosophila esterase 6

Esterase 6 fromDrosophila melanogaster is a carboxylesterase that belongs to the serine esterase multigene family. It has a basic histidine (His) at residue 187, adjacent to the reactive serine (Ser) at residue 188, whereas most other characterized members of the family have an acidic glutamate (Glu) in the equivalent position. We have used site-directedin vitro mutagenesis to replace the His codon of the esterase 6 gene with either Gln or Glu codons. The enzymes encoded by these active-site mutants and a wild-type control have been expressed, purified, and characterized. Substitution of Gln for His at position 187 has little effect on the biochemical properties of esterase 6, but the presence of Glu at this position is associated with three major differences. First, the pH optimum is increased from 7 to 9. Second, the mutant enzyme shows decreased activity for β-naphthyl esters andp-nitrophenyl acetate but has gained the ability to hydrolyze acetylthiocholine. Finally, the Gibb’s free energy of activation for the enzyme is increased. These results suggest that residue 187 interacts directly with the substrate alkyl group and that this interaction is fully realized in the transition state. We further propose that the presence of His rather than Glu at position 187 in esterase 6 contributes significantly to its functional divergence from the cholinesterases and that this divergence is due to different interactions between residue 187 and the substrate alkyl group.     

Exploring the active site cavity of human pancreatic lipase

Within the scope of improving the efficiency of pancreatic enzyme replacement therapy in cystic fibrosis, the feasibility of shifting the pH-activity profile of pancreatic lipase toward acidic values was investigated by site specific mutagenesis in different regions of the catalytic cavity. We have shown that introducing a negative charge close to the catalytic histidine induced a shift of the pH optimum toward acidic values but strongly reduced the lipase activity. On the other hand, a negative charge in the entrance of the catalytic cleft gives rise to a lipase with improved properties and twice more active than the native enzyme at acidic pH.     

X-ray structures of Aerococcus viridans lactate oxidase and its complex with D-lactate at pH 4.5 show an alpha-hydroxyacid oxidation mechanism

l-Lactate oxidase (LOX) belongs to a family of flavin mononucleotide (FMN)-dependent α-hydroxy acid-oxidizing enzymes. Previously, the crystal structure of LOX (pH 8.0) from Aerococcus viridans was solved, revealing that the active site residues are located around the FMN. Here, we solved the crystal structures of the same enzyme at pH 4.5 and its complex with d-lactate at pH 4.5, in an attempt to analyze the intermediate steps. In the complex structure, the d-lactate resides in the substrate-binding site, but interestingly, an active site base, His265, flips far away from the d-lactate, as compared with its conformation in the unbound state at pH 8.0. This movement probably results from the protonation of His265 during the crystallization at pH 4.5, because the same flip is observed in the structure of the unbound state at pH 4.5. Thus, the present structure appears to mimic an intermediate after His265 abstracts a proton from the substrate. The flip of His265 triggers a large structural rearrangement, creating a new hydrogen bonding network between His265-Asp174-Lys221 and, furthermore, brings molecular oxygen in between d-lactate and His265. This mimic of the ternary complex intermediate enzyme-substrate-O₂ could explain the reductive half-reaction mechanism to release pyruvate through hydride transfer. In the mechanism of the subsequent oxidative half-reaction, His265 flips back, pushing molecular oxygen into the substrate-binding site as the second substrate, and the reverse reaction takes place to produce hydrogen peroxide. During the reaction, the flip-flop action of His265 has a dual role as an active base/acid to define the major chemical steps. Our proposed reaction mechanism appears to be a common mechanistic strategy for this family of enzymes.     

Carboxypeptidase Y: structural basis for protein sorting and catalytic triad.

A yeast vacuolar protease, carboxypeptidase Y (CPY), is known to be involved in the C-terminal processing of peptides and proteins; however, its real function remains unclear. The CPY biosynthetic pathway has been used as a model system for protein sorting in eukaryotes. CPY is synthesized as a prepro-form that travels through the ER and Golgi to its final destination in vacuoles. In the course of studies on the transport mechanism of CPY, various post-translational events have been identified, e.g. carbohydrate modification and cleavage of the pre-segments. In addition, sorting signals and various sorting vehicles, similar to those found in higher eukaryotic cells, have been found. The catalytic triad in the active site of CPY makes this enzyme a serine protease. A unique feature distinguishing CPY from other serine proteases is its wide pH optimum, in particular its high activity at acidic pH. Several structural properties which might contribute to this unique feature exist such as a conserved free cysteine residue in the S1 substrate binding pocket, a recognition site for a C-terminal carboxyl group, and a disulfide zipper motif. The structural bases in CPY functions are discussed in this article.     

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase functions through a general acid-base catalyst pair mechanism

UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine deacetylase (LpxC) is a zinc-dependent enzyme that catalyzes the deacetylation of UDP-3-O-((R)-3-hydroxymyristoyl)-N-acetylglucosamine to form UDP-3-O-(R-hydroxymyristoyl)glucosamine and acetate. The structural similarity of the active site of LpxC to metalloproteases led to the proposal that LpxC functions via a metalloprotease-like mechanism. The pH dependence of k(cat)/Km catalyzed by Escherichia coli and Aquifex aeolicus LpxC displayed a bell-shaped curve (EcLpxC yields apparent pKa values of 6.4+/-0.1 and 9.1+/-0.1), demonstrating that at least two ionizations are important for maximal activity. Metal substitution and mutagenesis experiments suggest that the basic limb of the pH profile is because of deprotonation of a zinc-coordinated group such as the zinc-water molecule, whereas the acidic limb of the pH profile is caused by protonation of either Glu78 or His265. Furthermore, the magnitude of the activity decreases and synergy observed for the active site mutants suggest that Glu78 and His265 act as a general acid-base catalyst pair. Crystal structures of LpxC complexed with cacodylate or palmitate demonstrate that both Glu78 and His265 hydrogen-bond with the same oxygen atom of the tetrahedral intermediate and the product carboxylate. These structural features suggest that LpxC catalyzes deacetylation by using Glu78 and His265 as a general acid-base pair and the zinc-bound water as a nucleophile.     

Insights into the modulation of optimum pH by a single histidine residue in arginine deiminase from Pseudomonas aeruginosa

Abstract Arginine deiminase (ADI) is a potential antitumor agent for the arginine deprivation treatment of l-arginine auxotrophic tumors. The optimum pH of ADI varies significantly, yet little is known about the origin of this variety. Here, Pseudomonas aeruginosa ADI (PaADI), an enzyme that functions only at acidic pH, was utilized as the model system. The results of UV-pH titration imply that the nucleophilic Cys406 thiol group is protonated in the resting state. The H405R single mutation resulted in an altered pH optimum (from pH 5.5 to 6.5), an increased kcat (from 9.8 s-1 to 101.7 s-1 at pH 6.5), and a shifted pH rate dependence (ascending limb pKa from 3.6 to 4.4). Other mutants were constructed to investigate the effects of hydrogen bonding, charge distribution, and hydrophobicity on the properties of the enzyme. The pH optima of His405 mutants were all shifted to a relatively neutral pH except for the H405E mutant. The results of kinetic characterizations and molecular dynamic simulations revealed that the active site hydrogen bonding network involving Asp280 and His405 plays an important role in controlling the dependence of PaADI activity on pH. Moreover, the H405R variant showed increased cytotoxicity towards arginine auxotrophic cancer cell lines.     

Effect of a charged residue at the 213th site of thermolysin on the enzymatic activity

Considering the electrostatic potential of active site, four mutants of thermolysin (EC 3.4.24.4) are designed in an attempt to change the optimum pH of the hydrolytic activity toward acidic regions. On the basis of the numerical calculation of the electrostatic potential in the thermolysin molecule, Asp213 is targeted to be replaced by a basic residue, His, Lys, Arg or a neutral one, Asn. The mutant enzymes are produced inBacillus subtilis as a host using the method of site-directed mutagenesis and their optimum pH values for hydrolyzing a synthetic substrate furylacryloyl-Gly-l-Leu-NH₂ are found to be lowered by 0.2–0.4 pH units with reference to the wild type enzyme. The pl shifts of the mutants are evaluated. Neither optimum pH nor pl shift can be explained by the contribution of the pK change only at the mutation site. We find a clear negative correlation between the activities at pH 7.0 and the pI values among the four mutants and wild-type enzyme. It suggests that the contribution of pK shift of other residues must be taken into account in order to explain the activity change. Little change of thermal stability is observed among the mutants and wild type enzymes.     

The structure and unusual pH dependence of plastocyanin from the fern Dryopteris crassirhizoma. The protonation of an active site histidine is hindered by pi-pi interactions

Spectroscopic properties, amino acid sequence, electron transfer kinetics, and crystal structures of the oxidized (at 1.7 A resolution) and reduced form (at 1.8 A resolution) of a novel plastocyanin from the fern Dryopteris crassirhizoma are presented. Kinetic studies show that the reduced form of Dryopteris plastocyanin remains redox-active at low pH, under conditions where the oxidation of the reduced form of other plastocyanins is inhibited by the protonation of a solvent-exposed active site residue, His87 (equivalent to His90 in Dryopteris plastocyanin). The x-ray crystal structure analysis of Dryopteris plastocyanin reveals pi-pi stacking between Phe12 and His90, suggesting that the active site is uniquely protected against inactivation. Like higher plant plastocyanins, Dryopteris plastocyanin has an acidic patch, but this patch is located closer to the solvent-exposed active site His residue, and the total number of acidic residues is smaller. In the reactions of Dryopteris plastocyanin with inorganic redox reagents, the acidic patch (the "remote" site) and the hydrophobic patch surrounding His90 (the "adjacent" site) are equally efficient for electron transfer. These results indicate the significance of the lack of protonation at the active site of Dryopteris plastocyanin, the equivalence of the two electron transfer sites in this protein, and a possibility of obtaining a novel insight into the photosynthetic electron transfer system of the first vascular plant fern, including its molecular evolutionary aspects. This is the first report on the characterization of plastocyanin and the first three-dimensional protein structure from fern plant.     

Probing the catalytic mechanism of bovine pancreatic deoxyribonuclease I by chemical rescue

Previous structural and mutational studies of bovine pancreatic deoxyribonuclease I (bpDNase I) have demonstrated that the active site His134 and His252 played critical roles in catalysis. In our present study, mutations of these two His residues to Gln, Ala or Gly reduced the DNase activity by a factor of four to five orders of magnitude. When imidazole or primary amines were added exogenously to the Ala or Gly mutants, the residual DNase activities were substantially increased by 60-120-fold. The rescue with imidazole was pH- and concentration-dependent. The pH-activity profiles showed nearly bell-shaped curves, with the maximum activity enhancement for H134A at pH 6.0 and that for H252A at pH 7.5. These findings indicated that the protonated form of imidazole was responsible for the rescue in H134A, and the unprotonated form was for that in H252A, prompting us to assign unambiguously the roles for His134 as a general acid, and His252 as a general base, in bpDNase I catalysis.     

Electrostatic role of aromatic ring stacking in the pH-sensitive modulation of a chymotrypsin-type serine protease, Achromobacter protease I.

Achromobacter protease I (API) has a unique region of aromatic ring stacking with Trp169-His210 in close proximity to the catalytic triad. This paper reveals the electrostatic role of aromatic stacking in the shift in optimum pH to the alkaline region, which is the highest pH range (8.5-10) among chymotrypsin-type serine proteases. The pH-activity profile of API showed a sigmoidal distribution that appears at pH 8-10, with a shoulder at pH 6-8. Variants with smaller amino acid residues substituted for Trp169 had lower pH optima on the acidic side by 0-0.9 units. On the other hand, replacement of His210 by Ala or Ser lowered the acidic rim by 1.9 pH units, which is essentially identical to that of chymotrypsin and trypsin. Energy minimization for the mutant structures suggested that the side-chain of Trp169 stacked with His210 was responsible for isolation of the electrostatic interaction between His210 and the catalytic Asp113 from solvent. The aromatic stacking regulates the low activity at neutral pH and the high activity at alkaline pH due to the interference of the hydrogen bonded network in the catalytic triad residues.     

Histidine 440 controls the opening of colicin E1 channels in a lipid-dependent manner

The in vitro activity of many pore-forming toxins, in particular, the rate of increase in the membrane conductance induced by the channel-forming domain (P178) of colicin E1 is maximum at an acidic pH. However, after P178 binding at acidic conditions, a subsequent pH shift from 4 to 6 on both sides of the planar bilayer lipid membrane caused a large increase in the trans-membrane current which was solely due to an increase in the number of open channels. This effect required the presence of anionic lipid. Replacing the His440 residue of P178 by alanine eliminated the pH-shift effect thereby showing that it is associated with deprotonation of this histidine residue. It was concluded that alkalinization-induced weakening of the electrostatic interactions between colicin and the membrane surface facilitates conformational changes required for the transition of membrane-bound colicin molecules to an active channel state.     

Catalytic and structural role of a metal-free histidine residue in bovine Cu-Zn superoxide dismutase

Cu-Zn superoxide dismutase (SOD) contains a conserved, metal-free His residue at an opening of the backbone beta-barrel in addition to six Cu- and/or Zn-bound His residues in the active site. We examined the protonation and hydrogen bonding state of the metal-free His residue (His41) in bovine SOD by UV Raman spectroscopy. Analysis of the His Raman intensity at 1406 cm(-1) in a D2O solution has shown that His41 has a pKa of 9.4, consistent with the NMR and X-ray structures at acidic to neutral pH, in which two imidazole nitrogen atoms of cationic His41 are hydrogen bonded to the main chain C=O groups of Thr37 and His118. Upon deprotonation of His41 at pH 9.4, the Thr37-His41-His118 hydrogen bond bridge breaks on the His118 side and SOD loses 70% of its activity. Concomitantly, hydrogen-deuterium exchange is accelerated for amide groups of beta-strands, indicating an increased conformational fluctuation of the beta-barrel. Thr37 and His41 are in direct contact with Leu36, whose hydrophobic side chain closes off the opening of the beta-barrel, while His118 is indirectly connected to Arg141 that assists the docking of superoxide to Cu. These Raman findings strongly suggest that the His41-mediated hydrogen bond bridge plays a crucial role in keeping the protein structure suitable for highly efficient catalytic reactions. The catalytic and structural role of His41 is consistent with the observation that the mutation of His43 in human SOD (equivalent to His41 in bovine SOD) to Arg largely reduces the dismutase activity and the protein structural stability.     

Protein A-Mouse Acidic Mammalian Chitinase-V5-His Expressed in Periplasmic Space of Escherichia coli Possesses Chitinase Functions Comparable to CHO-Expressed Protein

Acidic mammalian chitinase (AMCase) has been shown to be associated with asthma in mouse models, allergic inflammation and food processing. Here, we describe an E. coli-expression system that allows for the periplasmic production of active AMCase fused to Protein A at the N-terminus and V5 epitope and (His)₆ tag (V5-His) at the C-terminus (Protein A-AMCase-V5-His) in E. coli. The mouse AMCase cDNA was cloned into the vector pEZZ18, which is an expression vector containing the Staphylococcus Protein A promoter, with the signal sequence and truncated form of Protein A for extracellular expression in E. coli. Most of the Protein A-AMCase-V5-His was present in the periplasmic space with chitinolytic activity, which was measured using a chromogenic substrate, 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside. The Protein A-AMCase-V5-His was purified from periplasmic fractions using an IgG Sepharose column followed by a Ni Sepharose chromatography. The recombinant protein showed a robust peak of activity with a maximum observed activity at pH 2.0, where an optimal temperature was 54°C. When this protein was preincubated between pH 1.0 and pH 11.0 on ice for 1 h, full chitinolytic activity was retained. This protein was also heat-stable till 54°C, both at pH 2.0 and 7.0. The chitinolytic activity of the recombinant AMCase against 4-nitrophenyl N,N′-diacetyl-β-D-chitobioside was comparable to the CHO-expressed AMCase. Furthermore, the recombinant AMCase bound to chitin beads, cleaved colloidal chitin and released mainly N,N′-diacetylchitobiose fragments. Thus, the E. coli-expressed Protein A-mouse AMCase-V5-His fusion protein possesses chitinase functions comparable to the CHO-expressed AMCase. This recombinant protein can be used to elucidate detailed biomedical functions of the mouse AMCase.