Catalytic Acid Residues Research Articles

The Role of a Loop in the Non-catalytic Domain B on the Hydrolysis/Transglycosylation Specificity of the 4-α-Glucanotransferase from Thermotoga maritima.

The mechanism by which glycoside hydrolases control the reaction specificity through hydrolysis or transglycosylation is a key element embedded in their chemical structures. The determinants of reaction specificity seem to be complex. We looked for structural differences in domain B between the 4-α-glucanotransferase from Thermotoga maritima (TmGTase) and the α-amylase from Thermotoga petrophila (TpAmylase) and found a longer loop in the former that extends towards the active site carrying a W residue at its tip. Based on these differences we constructed the variants W131G and the partial deletion of the loop at residues 120-124/128-131, which showed a 11.6 and 11.4-fold increased hydrolysis/transglycosylation (H/T) ratio relative to WT protein, respectively. These variants had a reduction in the maximum velocity of the transglycosylation reaction, while their affinity for maltose as the acceptor was not substantially affected. Molecular dynamics simulations allow us to rationalize the increase in H/T ratio in terms of the flexibility near the active site and the conformations of the catalytic acid residues and their associated pKas.

Key interactions convert amino acid side chains into strong acids and bases in the active sites of enzymes.

Enzymes catalyze reactions under mild conditions that might otherwise require extreme conditions such as high temperature or strong acid or base. To achieve this, amino acid side chains that are weak Brønsted acids or bases in the free amino acid become strong acids and bases in the active sites of many enzymes. Here we report on a set of 30 enzymes that represent all six major EC classes and a variety of different folds for which experimental studies of the mechanistic roles of the catalytic residues have been reported in the literature. Using macromolecular electrostatics techniques, it is shown that the catalytic aspartate and glutamate residues are strongly coupled to at least one other aspartate or glutamate residue, and often to multiple other carboxylate residues, with intrinsic pKa differences less than 1 pH unit. Sometimes these catalytic acidic residues are also coupled to a histidine residue, such that the intrinsic pKa of the acidic residue is higher than that of the histidine. Most catalytic lysine residues studied here are strongly coupled to tyrosine or cysteine residues, wherein the intrinsic pKa of the anion-forming residue is higher than that of the lysine. Some catalytic lysines are also coupled to other lysines with intrinsic pKa differences within 1 pH unit. Some general principles about the design of enzyme active sites are presented. These principles have significant implications for the engineering of novel enzymes. Supported by NSF CHE-1905214 (MJO).

Electrostatic Fingerprints of Catalytically Active Amino Acids in Enzymes

The computed electrostatic and proton transfer properties are studied for 20 enzymes that represent all six major EC classes and a variety of different folds. The properties of aspartate, glutamate, and lysine residues that have been previously experimentally determined to be catalytically active are reported. All catalytic aspartate and glutamate residues studied here are strongly coupled to at least one other aspartate or glutamate residue and often to multiple other carboxylate residues with intrinsic pKa differences less than 1 pH unit. Sometimes these catalytic acidic residues are also coupled to a histidine residue, such that the intrinsic pKa of the acidic residue is higher than that of the histidine. All catalytic lysine residues studied here are strongly coupled to tyrosine or cysteine residues, wherein the intrinsic pKa of the anion‐forming residue is higher than that of the lysine. Some catalytic lysines are also coupled to other lysines with intrinsic pKa differences within 1 pH unit. Some evidence of the possible types of interactions that facilitate nucleophilicity is discussed. The interactions reported here provide important clues about how side chain functional groups that are weak Br⊘nsted acids or bases for the free amino acid in solution can become strong acids, bases or nucleophiles in the enzymatic environment. Such interactions are important considerations in enzyme design.

Electrostatic fingerprints of catalytically active amino acids in enzymes.

The computed electrostatic and proton transfer properties are studied for 20 enzymes that represent all six major enzyme commission classes and a variety of different folds. The properties of aspartate, glutamate, and lysine residues that have been previously experimentally determined to be catalytically active are reported. The catalytic aspartate and glutamate residues studied here are strongly coupled to at least one other aspartate or glutamate residue and often to multiple other carboxylate residues with intrinsic pKa differences less than 1 pH unit. Sometimes these catalytic acidic residues are also coupled to a histidine residue, such that the intrinsic pKa of the acidic residue is higher than that of the histidine. All catalytic lysine residues studied here are strongly coupled to tyrosine or cysteine residues, wherein the intrinsic pKa of the anion-forming residue is higher than that of the lysine. Some catalytic lysines are also coupled to other lysines with intrinsic pKa differences within 1 pH unit. Some evidence of the possible types of interactions that facilitate nucleophilicity is discussed. The interactions reported here provide important clues about how side chain functional groups that are weak Brønsted acids or bases for the free amino acid in solution can achieve catalytic potency and become strong acids, bases or nucleophiles in the enzymatic environment.

Systematic virtual screening in search of SARS CoV-2 inhibitors against spike glycoprotein: pharmacophore screening, molecular docking, ADMET analysis and MD simulations.

In the absence of efficient anti-viral medications, the coronavirus disease 2019 (COVID-19), stemming from severe acute respiratory syndrome coronavirus-2 (SARS CoV-2), has spawned a worldwide catastrophe and global emergency. Amidst several anti-viral targets of COVID-19, spike glycoprotein has been recognized as an essential target for the viral entry into the host cell. In the search of effective SARS CoV-2 inhibitors acting against spike glycoprotein, the virtual screening of 175,851 ligands from the 2020.1 Asinex BioDesign library has been performed using in silico tools like SiteMap analysis, pharmacophore-based screening, molecular docking using different levels of precision, such as high throughput virtual screening, standard precision and extra precision, followed by absorption, distribution, metabolism, excretion and toxicity analysis, and molecular dynamics (MD) simulation. Following a molecular docking study, seventeen molecules (with a docking score of less than − 6.0) were identified having the substantial interactions with the catalytic amino acid and nucleic acid residues of spike glycoprotein at the binding site. In investigations using MD simulations for 10 ns, the hit molecules (1 and 2) showed adequate compactness and uniqueness, as well as satisfactory stability. These computational research findings have offered a key starting point in the field of design and development of novel SARS CoV-2 entry inhibitors with appropriate drug likeliness.Graphical abstract Supplementary InformationThe online version contains supplementary material available at 10.1007/s11030-022-10394-9.

The Structurally Unusual Retaining β‐Kdo Glycosyltransferase WbbB Uses a Double‐Displacement Mechanism with an Intermediate Adduct Rearrangement Step

WbbB is a modular, trifunctional glycosyltransferase (GT) which synthesizes lipopolysaccharide O‐antigen in Raoultella terrigena. The two C‐terminal WbbB GT domains build the polysaccharide repeat, while the N‐terminal GT domain acts as a terminator, adding a β‐Kdo (3‐deoxy‐d‐manno‐oct‐2‐ulosonic acid) residue to O3 to rhamnose as required for O‐antigen export. This N‐terminal GT domain is highly unusual in sequence and was not recognized as a GT by bioinformatics tools. The structure shows the dual Rossmann‐fold motifs characteristic of GT‐Bs, but with extensive deletions, insertions and rearrangements result in a unique architecture. The CMP‐binding site, however, retains motifs homologous to GT‐B sialyltransferases. WbbB is a retaining GT, transferring Kdo from the CMP‐®‐Kdo donor with net retention of anomeric configuration. All well‐characterized retaining GTs seem to employ a front‐side SNi substitution mechanism. This is somewhat surprising, as analogous retaining glycosyl hydrolases use a double‐displacement mechanism; here an essential catalytic acid residue attacks the donor saccharide to form a covalently bonded intermediate, with a second acidic residue acting as a general base to hydrolyse this adduct. We show, using mass spectrometry, that WbbB forms a Kdo adduct with Asp232 and its variants (D232N or D232C), while a D232A variant is both wholly inactive and forms no adduct. The x‐ray structure of D232N CMP‐®‐Kdo donor complex shows that the anomeric carbon of the donor is inaccessible to any potential acceptor but is instead positioned immediately in contact with, and in line with, the carboxylate of Asp232. Structures of D232N‐ and D232C‐Kdo adducts show that the Kdo adduct is rearranged into a second half‐site, interacting with a distinct set of catalytic residues. A ternary complex of D232C‐Kdo plus a synthetic acceptor shows that Glu158 forms direct hydrogen bonds with O3 of rhamnose, positioned adjacent to and in‐line with the anomeric carbon of Kdo, activating it for attack. Glu158 is also essential, with variants being wholly inactive. Together, this shows that WbbB uses a glycosyl hydrolase‐like double‐displacement mechanism. We propose that crowding around the anomeric carbon by the carboxylate group likely precludes retaining ulosonic acid transferases using the more straightforward SNi mechanism, while adduct rearrangement avoids having the acceptor compete with the leaving nucleotide for access to the anomeric carbon of the donor.Support or Funding InformationThis work was funded by an NSERC Discovery grant (04045‐2015) to MSK and support from the Canadian Glycomics Network (SD‐1) to TLL; TJBF is the recipient of a University of Guelph Graduate Excellence Entrance Scholarship and an Ontario Graduate Scholarship.

Structural and biochemical studies of the glucuronoyl esterase OtCE15A illuminate its interaction with lignocellulosic components

Glucuronoyl esterases (GEs) catalyze the cleavage of ester linkages between lignin and glucuronic acid moieties on glucuronoxylan in plant biomass. As such, GEs represent promising biochemical tools in industrial processing of these recalcitrant resources. However, details on how GEs interact and catalyze degradation of their natural substrates are sparse, calling for thorough enzyme structure-function studies. Presented here is a structural and mechanistic investigation of the bacterial GE OtCE15A. GEs belong to the carbohydrate esterase family 15 (CE15), which is in turn part of the larger α/β-hydrolase superfamily. GEs contain a Ser-His-Asp/Glu catalytic triad, but the location of the catalytic acid in GEs has been shown to be variable, and OtCE15A possesses two putative catalytic acidic residues in the active site. Through site-directed mutagenesis, we demonstrate that these residues are functionally redundant, possibly indicating the evolutionary route toward new functionalities within the family. Structures determined with glucuronate, in both native and covalently bound intermediate states, and galacturonate provide insights into the catalytic mechanism of CE15. A structure of OtCE15A with the glucuronoxylooligosaccharide 23-(4-O-methyl-α-d-glucuronyl)-xylotriose (commonly referred to as XUX) shows that the enzyme can indeed interact with polysaccharides from the plant cell wall, and an additional structure with the disaccharide xylobiose revealed a surface binding site that could possibly indicate a recognition mechanism for long glucuronoxylan chains. Collectively, the results indicate that OtCE15A, and likely most of the CE15 family, can utilize esters of glucuronoxylooligosaccharides and support the proposal that these enzymes work on lignin-carbohydrate complexes in plant biomass.

Structure and biochemical characterization of glucose tolerant β-1,4 glucosidase (HtBgl) of family 1 glycoside hydrolase from Hungateiclostridium thermocellum

β-1,4-glucosidase (HtBgl) of family 1 glycoside hydrolase from Hungateiclostridium thermocellum was cloned in pET28a(+) vector, expressed, biochemically and structurally characterized. HtBgl displayed 67 U/mg activity against 4-nitrophenyl-β-d-glucopyranoside, followed by 180 U/mg against cellobiose and 42 U/mg activity against 4-nitrophenyl-β-d-galactopyranoside. HtBgl displayed an optimum temperature of 65 °C and an optimum pH of 6.0. HtBgl was stable in the pH range, 4.0–8.0 and displayed the thermostability up to 60 °C for 1 h. HtBgl displayed the glucose tolerance up to 750 mM and retained ~70% activity after 20 h. HtBgl crystal structure submitted (PDB id 5OGZ) by others exhibited a classical Triosephosphate Isomerase, (β/α)8-barrel fold. Protein melting analysis of HtBgl exhibited a single peak at 78 °C and the addition of 5 mM Mg2+ shifted the peak to 82 °C. Molecular dynamics studies showed that the amino acid residues from 351 to 375 exhibit the flexibility due to the presence of the catalytic acid residue. The structure comparison of HtBgl with homologous proteins and its docking analysis with probable ligands revealed that the residues, E166 and E355 are involved in the catalysis. The SAXS analysis of HtBgl showed that the protein is monomeric and present in a fully folded state. The radius of gyration (Rg) found was 2.15–2.26 nm. The bell-shaped curve obtained by Kratky plot analysis displayed the globular shape and fully folded state with flexibility in the N-terminal region. The HtBgl crystal structure superposed well with the SAXS derived dummy atom model.

Gluco-1 H-imidazole: A New Class of Azole-Type β-Glucosidase Inhibitor.

Gluco-azoles competitively inhibit glucosidases by transition-state mimicry and their ability to interact with catalytic acid residues in glucosidase active sites. We noted that no azole-type inhibitors described, to date, possess a protic nitrogen characteristic for 1H-imidazoles. Here, we present gluco-1H-imidazole, a gluco-azole bearing a 1H-imidazole fused to a glucopyranose-configured cyclitol core, and three close analogues as new glucosidase inhibitors. All compounds inhibit human retaining β-glucosidase, GBA1, with the most potent ones inhibiting this enzyme (deficient in Gaucher disease) on a par with glucoimidazole. None inhibit glucosylceramide synthase, cytosolic β-glucosidase GBA2 or α-glucosidase GAA. Structural, physical and computational studies provide first insights into the binding mode of this conceptually new class of retaining β-glucosidase inhibitors.

Alpha/beta-hydrolases: A unique structural motif coordinates catalytic acid residue in 40 protein fold families.

The alpha/beta-hydrolases are a family of acid-base-nucleophile catalytic triad enzymes with a common fold, but using a wide variety of substrates, having different pH optima, catalyzing unique catalytic reactions and often showing improved chemical and thermo stability. The ABH enzymes are prime targets for protein engineering. Here, we have classified active sites from 51 representative members of 40 structural ABH fold families into eight distinct conserved geometries. We demonstrate the occurrence of a common structural motif, the catalytic acid zone, at the catalytic triad acid turn. We show that binding of an external ligand does not change the structure of the catalytic acid zone and both the ligand-free and ligand-bound forms of the protein belong to the same catalytic acid zone subgroup. We also show that the catalytic acid zone coordinates the position of the catalytic histidine loop directly above its plane, and consequently, fixes the catalytic histidine in a proper position near the catalytic acid. Finally, we demonstrate that the catalytic acid zone plays a key role in multi-subunit complex formation in ABH enzymes, and is involved in interactions with other proteins. As a result, we speculate that each of the catalytic triad residues has its own supporting structural scaffold, similar to the catalytic acid zone, described above, which together form the extended catalytic triad motif. Each scaffold coordinates the function of its respective catalytic residue, and can even compensate for the loss of protein function, if the catalytic amino acid is mutated.

Crystal structure and biological implications of a glycoside hydrolase family 55 β-1,3-glucanase from Chaetomium thermophilum

Crystal structures of a β-1,3-glucanase from the thermophilic fungus Chaetomium thermophilum were determined at 1.20 and 1.42Å resolution in the free and glucose-bound form, respectively. This is the third structure of a family 55 glycoside hydrolase (GH55) member and the second from a fungus. Based on comparative structural studies and site-directed mutagenesis, Glu654 is proposed as the catalytic acid residue. The substrate binding cleft exhibits restricted access on one side, rendering the enzyme as an exo-β-1,3-glucanase as confirmed also by thin layer chromatography experiments. A lack of stacking interactions was found at the substrate binding cleft, suggesting that interactions at positions −1, +1 and +2 are sufficient to orientate the substrate. A binding pocket was identified that could explain binding of branched laminarin and accumulation of laminaritriose.

Site-directed mutagenesis of α-L-rhamnosidase from Alternaria sp. L1 to enhance synthesis yield of reverse hydrolysis based on rational design.

The α-L-rhamnosidase catalyzes the hydrolytic release of rhamnose from polysaccharides and glycosides and is widely used due to its applications in a variety of industrial processes. Our previous work reported that a wild-type α-L-rhamnosidase (RhaL1) from Alternaria sp. L1 could synthesize rhamnose-containing chemicals (RCCs) though reverse hydrolysis reaction with inexpensive rhamnose as glycosyl donor. To enhance the yield of reverse hydrolysis reaction and to determine the amino acid residues essential for the catalytic activity of RhaL1, site-directed mutagenesis of 11 residues was performed in this study. Through rationally designed mutations, the critical amino acid residues which may form direct or solvent-mediated hydrogen bonds with donor rhamnose (Asp252, Asp257, Asp264, Glu530, Arg548, His553, and Trp555) and may form the hydrophobic pocket in stabilizing donor (Trp261, Tyr302, Tyr316, and Trp369) in active-site of RhaL1 were analyzed, and three positive mutants (W261Y, Y302F, and Y316F) with improved product yield stood out. From the three positive variants, mutant W261Y accelerated the reverse hydrolysis with a prominent increase (43.7%) in relative yield compared to the wild-type enzyme. Based on the 3D structural modeling, we supposed that the improved yield of mutant W261Y is due to the adjustment of the spatial position of the putative catalytic acid residue Asp257. Mutant W261Y also exhibited a shift in the pH-activity profile in hydrolysis reaction, indicating that introducing of a polar residue in the active site cavity may affect the catalysis behavior of the enzyme.

Characteristics, protein engineering and applications of microbial thermostable pullulanases and pullulan hydrolases.

Pullulan hydrolyzing enzymes are endoacting, classified based on the substrate specificity and hydrolysis products as pullulanases (type I and II) and pullulan hydrolases (type I, II and III). Pullulanases and pullulan hydrolase type I are produced by bacteria and archaea. Among bacteria, many mesophilic, thermophilic and hyperthermophilic bacteria produce pullulanases and neopullulanases. While pullulan hydrolase type II and type III are produced by fungi and archaea, respectively. These are multi-domain proteins with three conserved catalytic acidic residues of the glycosyl hydrolases. The recent advances in molecular biology and protein engineering via mutagenesis and truncation led to improvement in thermostability, catalytic activity and substrate specificity. Pullulanases are debranching enzymes, which are widely employed in starch saccharification that minimizes the use of glucoamylase (approx. 50%) and reduces the total reaction time of the industrial starch conversion process. The thermostable amylopullulanases are useful in one-step starch liquefaction and saccharification, which replaces amylolytic enzymes like α-amylase and glucoamylase, thus resulting in the reduction in the cost of sugar production. The enzymes also find application in making resistant starches and as an antistale in bread making. Panose and isopanose containing syrups are useful as prebiotics, while panose has also been reported to display anticarcinogenic activity. This review focuses on the distinguishing features of these enzymes based on the analysis of amino acid sequences and domain structure, besides highlighting recent advances in the molecular biology and protein engineering for enhancing their thermostability, catalytic activity and substrate specificity. This review also briefly summarizes the potential applications of pullulanases and pullulan hydrolases.

Direct determination of protonation states and visualization of hydrogen bonding in a glycoside hydrolase with neutron crystallography

Glycoside hydrolase (GH) enzymes apply acid/base chemistry to catalyze the decomposition of complex carbohydrates. These ubiquitous enzymes accept protons from solvent and donate them to substrates at close to neutral pH by modulating the pKa values of key side chains during catalysis. However, it is not known how the catalytic acid residue acquires a proton and transfers it efficiently to the substrate. To better understand GH chemistry, we used macromolecular neutron crystallography to directly determine protonation and ionization states of the active site residues of a family 11 GH at multiple pD (pD=pH+0.4) values. The general acid glutamate (Glu) cycles between two conformations, upward and downward, but is protonated only in the downward orientation. We performed continuum electrostatics calculations to estimate the pKa values of the catalytic Glu residues in both the apo- and substrate-bound states of the enzyme. The calculated pKa of the Glu increases substantially when the side chain moves down. The energy barrier required to rotate the catalytic Glu residue back to the upward conformation, where it can protonate the glycosidic oxygen of the substrate, is 4.3 kcal/mol according to free energy simulations. These findings shed light on the initial stage of the glycoside hydrolysis reaction in which molecular motion enables the general acid catalyst to obtain a proton from the bulk solvent and deliver it to the glycosidic oxygen.

Modulating the pH−Activity Profile of Cellulase by Substitution: Replacing the General Base Catalyst Aspartate with Cysteinesulfinate in Cellulase A from Cellulomonas fimi

Cellulase A (CenA) from Cellulomonas fimi is an inverting glycoside hydrolase and a member of family 6 of the CAZy database classification system. We replaced its putative catalytic base aspartyl residues, Aps392 and Asp216, with cysteinesulfinate using a combination of site-directed mutagenesis and chemical modification to investigate the applicability of this approach for the modulation of enzymatic properties. The substituted cysteinyl residues were oxidized to cysteinesulfinic acid with hydrogen peroxide, and the resulting protein products were demonstrated to retain their native structure. Oxidation of the Asp392Cys mutant enzyme restored 52% of wild-type activity when assessed at pH 7.5, whereas Asp216Cys CenA remained inactive. This suggests that Asp216 is not the catalytic base and provides further support for Asp392 performing this role. Similar substitution of the catalytic acid residue Asp252 or the catalytic nucleophile of the retaining enzyme Cel5A from Thermobifida fusca failed to produce active enzymes. This indicates a potential utility of this approach for uniquely identifying catalytic base residues. The replacement of Asp392 with cysteinesulfinate induced an acidic shift in the pH profile of the enzyme such that this enzyme derivative was more active than wild-type CenA below pH 5.5. These data demonstrate the potential of combining site-directed mutagenesis with chemical modification as a viable approach for the modulation of cellulases, and potentially other glycoside hydrolases, at low pH.

Structure and Mechanism of the Lincosamide Antibiotic Adenylyltransferase LinB

Lincosamides make up an important class of antibiotics used against a wide range of pathogens, including methicillin-resistant Staphylococcus aureus. Predictably, lincosamide-resistant microorganisms have emerged with antibiotic modification as one of their major resistance strategies. Inactivating enzymes LinB/A catalyze adenylylation of the drug; however, little is known about their mechanistic and structural properties. We determined two X-ray structures of LinB: ternary substrate- and binary product-bound complexes. Structural and kinetic characterization of LinB, mutagenesis, solvent isotope effect, and product inhibition studies are consistent with a mechanism involving direct in-line nucleotidyl transfer. The characterization of LinB enabled its classification as a member of a nucleotidyltransferase superfamily, along with nucleotide polymerases and aminoglycoside nucleotidyltransferases, and this relationship offers further support for the LinB mechanism. The LinB structure provides an evolutionary link to ancient nucleotide polymerases and suggests that, like protein kinases and acetyltransferases, these are proto-resistance elements from which drug resistance can evolve.

The absence of an identifiable single catalytic base residue in Thermobifida fusca exocellulase Cel6B

Thermobifida fusca exocellulase Cel6B acts by an inverting hydrolysis mechanism; however, the catalytic acid and base residues for this enzyme have not been confirmed. Site-directed mutagenesis and kinetic studies were used to show that Asp274 is the catalytic acid, which is consistent with what is found for other members of family-6 glycoside hydrolases; however, a single catalytic base was not identified. Mutation of all putative catalytic base residues, within 6 A of the -1/+1 glucose subsites, including the highly conserved Asp226, Asp497 and Glu495, as well as Ser232 and Tyr220, did not reveal a catalytic base, although these residues are all important for activity. We propose a novel hydrolysis mechanism for T. fusca Cel6B involving a proton-transferring network to carry out the catalytic base function.

Mechanism of Cdc25B Phosphatase with the Small Molecule Substrate p-Nitrophenyl Phosphate from QM/MM-MFEP Calculations

Cdc25B is a dual-specificity phosphatase that catalyzes the dephosphorylation of the Cdk2/CycA protein complex. This enzyme is an important regulator of the human cell cycle and has been identified as a potential anticancer target. In general, protein tyrosine phosphatases are thought to bind the dianionic form of the phosphate and employ general acid catalysis via the Asp residue in the highly conserved WPD-loop. However, the Cdc25 phosphatases form a special subfamily based on their distinct differences from other protein tyrosine phosphatases. Although Cdc25B contains the (H/V)CX(5)R catalytic motif present in all other protein tyrosine phosphatases, it lacks an analogous catalytic acid residue. No crystallographic data currently exist for the complex of Cdc25B with Cdk2/CycA, so in addition to its natural protein substrate, experimental and theoretical studies are often carried out with small molecule substrates. In an effort to gain understanding of the dephosphorylation mechanism of Cdc25B with a commonly used small molecule substrate, we have performed simulations of the rate-limiting step of the reaction catalyzed by Cdc25B with the substrate p-nitrophenyl phosphate using the recently developed QM/MM Minimum Free Energy Path method (Hu et al. J. Chem. Phys. 2008, 034105). We have simulated the first step of the reaction with both the monoanionic and the dianionic forms of the substrate, and our calculations favor a mechanism involving the monoanionic form. Thus, Cdc25 may employ a unique dephosphorylation mechanism among protein tyrosine phosphatases, at least in the case of the small molecule substrate p-nitrophenyl phosphate.

Mutagenesis and Modeling of the Peroxiredoxin (Prx) Complex with the NMR Structure of ATP-Bound Human Sulfiredoxin Implicate Aspartate 187 of Prx I as the Catalytic Residue in ATP Hydrolysis

The catalytic cysteine of certain members of the peroxiredoxin (Prx) family can be hyperoxidized to cysteinesulfinic acid during reduction of peroxides. Sulfiredoxin is responsible for the ATP-dependent reduction of cysteinesulfinic acid (SO2H) of hyperoxidized Prx. Here we report the NMR solution structure of human sulfiredoxin (hSrx), both with and without bound ATP, and we model the complex of ATP-bound hSrx with Prx. Binding ATP causes only small changes in the NMR structure of hSrx, and the bound ATP conformation is quite similar to that seen for the previously reported X-ray structure of the ADP-hSrx complex. Although hSrx binds ATP, it does not catalyze hydrolysis by itself and has no catalytic acid residue typical of most ATPase and kinase family proteins. For modeling the complex, the ATP-bound hSrx was docked to hyperoxidized Prx II using EMAP of CHARMM. In the model complex, Asn186 of Prx II (Asp187 of Prx I) is in contact with the hSrx-bound ATP beta- and gamma-phosphate groups. Asp187 of Prx I was mutated to alanine and asparagine, and binding and activity of the mutants with hSrx were compared to those of the wild type. For the D187N mutant, both binding and hydrolysis and reduction activities were comparable to those of the wild type, whereas for D187A, binding was unimpaired but ATP hydrolysis and reduction did not occur. The modeling and mutagenesis analyses strongly implicate Asp187 of Prx I as the catalytic residue responsible for ATP hydrolysis in the cysteinesulfinic acid reduction of Prx by hSrx.

Role of metal ions in catalysis by HIV integrase analyzed using a quantitative PCR disintegration assay

Paired metal ions have been proposed to be central to the catalytic mechanisms of RNase H nucleases, bacterial transposases, Holliday junction resolvases, retroviral integrases and many other enzymes. Here we present a sensitive assay for DNA transesterification in which catalysis by human immunodeficiency virus-type 1 (HIV-1) integrase (IN) connects two DNA strands (disintegration reaction), allowing detection using quantitative PCR (qPCR). We present evidence suggesting that the three acidic residues of the IN active site function through metal binding using metal rescue. In this method, the catalytic acidic residues were each substituted with cysteines. Mn2+ binds tightly to the sulfur atoms of the cysteine residues, but Mg2+ does not. We found that Mn2+, but not Mg2+, could rescue catalysis of each cysteine-substituted enzyme, providing evidence for functionally important metal binding by all three residues. We also used the PCR-boosted assay to show that HIV-1 IN could carry out transesterification reactions involving DNA 5′ hydroxyl groups as well as 3′ hydroxyls as nucleophiles. Lastly, we show that Mn2+ by itself (i.e. without enzyme) can catalyze formation of a low level of PCR-amplifiable product under extreme conditions, allowing us to estimate the rate enhancement due to the IN-protein scaffold as at least 60 million-fold.