Oxocarbenium Ion-like Transition State Research Articles

A metabolic inhibitor blocks cellular fucosylation and enables production of afucosylated antibodies

The fucosylation of glycoproteins regulates diverse physiological processes. Inhibitors that can control cellular levels of protein fucosylation have consequently emerged as being of high interest. One area where inhibitors of fucosylation have gained significant attention is in the production of afucosylated antibodies, which exhibit superior antibody-dependent cell cytotoxicity as compared to their fucosylated counterparts. Here, we describe β-carbafucose, a fucose derivative in which the endocyclic ring oxygen is replaced by a methylene group, and show that it acts as a potent metabolic inhibitor within cells to antagonize protein fucosylation. β-carbafucose is assimilated by the fucose salvage pathway to form GDP-carbafucose which, due to its being unable to form the oxocarbenium ion-like transition states used by fucosyltransferases, is an incompetent substrate for these enzymes. β-carbafucose treatment of a CHO cell line used for high-level production of the therapeutic antibody Herceptin leads to dose-dependent reductions in core fucosylation without affecting cell growth or antibody production. Mass spectrometry analyses of the intact antibody and N-glycans show that β-carbafucose is not incorporated into the antibody N-glycans at detectable levels. We expect that β-carbafucose will serve as a useful research tool for the community and may find immediate application for the rapid production of afucosylated antibodies for therapeutic purposes.

Mechanistic Understanding of Ring-Opening of Tetrahydrofurfuryl Alcohol over WOx-Modified Pt Model Surfaces and Powder Catalysts

The upgrading of heterocyclic biomass-derived oxygenates such as tetrahydrofurfuryl alcohol (THFA) via ring-opening is a promising pathway to produce value-added diol molecules using renewable carbon sources. This study combines model surface experiments, first-principles calculations, and powder catalyst characterization and activity evaluation to unravel the nature of the Pt and WOx active sites and the reaction mechanism of the THFA ring-opening reaction on a WOx/Pt inverse oxide catalyst. Temperature-programmed desorption (TPD) and high-resolution electron energy loss spectroscopy (HREELS) measurements on model surfaces demonstrated that THFA ring opened on Pt(111) but underwent further decomposition due to its strong bonding with the surface. However, WOx deposited on Pt(111) altered the interaction strength between the ring-opened intermediate and the surface to a proper extent to facilitate the facile desorption of the desired 1,5-pentanediol (1,5-PeD) product. Density functional theory (DFT) calculations showed that WOx/Pt(111) could promote ring opening of THFA via an oxocarbenium ion-like transition state, which was stabilized by hydrogen bonding with the hydroxyl groups of WOx. The hydrogenation of the ring-opened 5-hydroxyvaleraldehyde intermediate to 1,5-PeD was then feasible via Brønsted acid sites present on WOx. Steady-state activity studies on the corresponding powder catalysts showed that the 1,5-PeD selectivity increased from 20% on Pt/SiO2 to 65% on WOx/Pt/SiO2 with 1 wt % WOx loading, consistent with model surface experiments and DFT calculations. This study demonstrates the feasibility of using model surface experiments and first-principles calculations to guide practical catalyst design, and provides a design strategy that can be applied to the selective ring-opening of relevant heterocyclic biomass-derived oxygenates.

Side Chain Conformation Restriction in the Catalysis of Glycosidic Bond Formation by Leloir Glycosyltransferases, Glycoside Phosphorylases, and Transglycosidases.

Carbohydrate side chain conformation is an important factor in the control of reactivity at the anomeric center, ie, in the making and breaking of glycosidic bonds, whether chemically or, for hydrolysis, by glycoside hydrolases. In nature glycosidic bond formation is catalyzed out by glycosyltransferases (GTs), glycoside phosphoryases, and transglycosidases. By analysis of 118 crystal structures of sugar nucleotide dependent (Leloir) GTs, 136 crystal structures of glycoside phosphorylases, and 54 crystal structures of transglycosidases bound to hexopyranosides or their analogs at the donor site (-1 site), we determined that most enzymes that catalyze glycoside synthesis, be they GTs, glycoside phosphorylases or transglycosidases, restrict their substrate side chains to the most reactive gauche,gauche (gg) conformation to achieve maximum stabilization of the oxocarbenium ion-like transition state for glycosyl transfer. The galactose series deviates from this trend, with α-galactosyltransferases preferentially restricting their substrates to the second-most reactive gauche,trans (gt) conformation, and β-galactosyltransferases favoring the least reactive trans,gauche (tg) conformation. This insight will help progress the design and development of improved, conformationally-restricted GT inhibitors that take advantage of these inherent side chain preferences.

An Epoxide Intermediate in Glycosidase Catalysis.

Retaining glycoside hydrolases cleave their substrates through stereochemical retention at the anomeric position. Typically, this involves two-step mechanisms using either an enzymatic nucleophile via a covalent glycosyl enzyme intermediate or neighboring-group participation by a substrate-borne 2-acetamido neighboring group via an oxazoline intermediate; no enzymatic mechanism with participation of the sugar 2-hydroxyl has been reported. Here, we detail structural, computational, and kinetic evidence for neighboring-group participation by a mannose 2-hydroxyl in glycoside hydrolase family 99 endo-α-1,2-mannanases. We present a series of crystallographic snapshots of key species along the reaction coordinate: a Michaelis complex with a tetrasaccharide substrate; complexes with intermediate mimics, a sugar-shaped cyclitol β-1,2-aziridine and β-1,2-epoxide; and a product complex. The 1,2-epoxide intermediate mimic displayed hydrolytic and transfer reactivity analogous to that expected for the 1,2-anhydro sugar intermediate supporting its catalytic equivalence. Quantum mechanics/molecular mechanics modeling of the reaction coordinate predicted a reaction pathway through a 1,2-anhydro sugar via a transition state in an unusual flattened, envelope (E3) conformation. Kinetic isotope effects (kcat/KM) for anomeric-2H and anomeric-13C support an oxocarbenium ion-like transition state, and that for C2-18O (1.052 ± 0.006) directly implicates nucleophilic participation by the C2-hydroxyl. Collectively, these data substantiate this unprecedented and long-imagined enzymatic mechanism.

Structural insight into molecular mechanism of cytokinin activating protein from Pseudomonas aeruginosa PAO1.

Cytokinin (CK)-activating enzyme, called LOG, is a phosphoribohydrolase that hydrolyzes nucleotides into nucleobases and phosphoriboses. This reaction is a fascinating target for regulation of cellular active CK. However, misannotation of LOG as a lysine decarboxylase and the lack of detailed catalytic and substrate-binding mechanisms have prevented studies of LOG at a protein-level. In this study, we determined the crystal structure of PA4923 from Pseudomonas aeruginosa PAO1. The overall structure of PA4923 resembles those of type-I LOGs, and it exhibited phosphoribohydrolase activity against AMP. These observations indicated that PA4923 functions as an LOG. We also determined the PaLOG structure in complex with AMP and elucidated the detailed binding mode of LOG against the AMP substrate. Interestingly, PaLOG undergoes an open/closed conformational change upon binding AMP, during which the Glu74 residue located on the β3-β4 connecting loop flips 180° and moves 13 Å towards the AMP molecule. Structural and amino acid sequence comparisons of LOGs suggest that this conformational change upon substrate binding might be a common phenomenon in LOGs. In addition, based on our structural studies and the reported catalytic mechanism of nucleoside hydrolases, we proposed a catalytic mechanism for LOG in which an oxocarbenium ion-like transition state is formed.

Mechanistic Pathway on Human α-Glucosidase Maltase-Glucoamylase Unveiled by QM/MM Calculations.

The excessive consumption of starch in human diets is associated with highly prevalent chronic metabolic diseases such as type 2 diabetes and obesity. α-Glucosidase enzymes contribute to the digestion of starch into glucose and are thus attractive therapeutic targets for diabetes. Given that the active sites of the various families of α-glucosidases have different sizes and structural features, atomistic descriptions of the catalytic mechanisms of these enzymes can support the development of potent and selective new inhibitors. Maltase-glucoamylase (MGAM), in particular, has a N-terminal catalytic domain (NtMGAM) that has shown high inhibitor selectivity. We provide here the first theoretical study of the human NtMGAM catalytic domain, employing a hybrid QM/MM approach with the ONIOM method to disclose the full atomistic details of the reactions promoted by this domain. We observed that the catalytic activity follows the classical Koshland double-displacement mechanistic pathway that uses general acid and base catalysts. A covalent glycosyl-enzyme intermediate was formed and hydrolyzed in the first and second mechanistic steps, respectively, through oxocarbenium ion-like transition state structures. The overall reaction is of dissociative type. Both transition state geometries differ from those known to occur in other glycosidases. The activation free energy for the glycosylation rate-limiting step agrees with the experimental barrier of 15.8 kcal·mol-1. Both individual mechanistic steps of the reaction are exoergonic. These structural results may serve as the basis for the design of transition state analogue inhibitors that specifically target the intestinal NtMGAM catalytic domain, thus delaying the production of glucose in diabetic and obese patients.

Enzymatic C-glycosylation: Insights from the study of a complementary pair of plant O- and C-glucosyltransferases

C-Glycosylation presents a rare mode of sugar attachment to the core structure of natural products and is catalyzed by a special type of Leloir C-glycosyltransferases (C-GTs). Elucidation of mechanistic principles for these glycosyltransferases (GTs) is of fundamental interest, and it could also contribute to the development of new biocatalysts for the synthesis of valuable C-glycosides, potentially serving as analogues of the highly hydrolysis-sensitive O‑glycosides. Enzymatic glucosylation of the natural dihydrochalcone phloretin from UDP‑D-glucose was applied as a model reaction in the study of a structurally and functionally homologous pair of plant glucosyltransferases, where the enzyme from rice (Oryza sativa) was specific for C-glycosylation and the enzyme from pear (Pyrus communis) was specific for O-glycosylation. We show that distinct active-site motifs are used by the two enzymes to differentiate between C- and O-glucosylation of the phloretin acceptor. An enzyme design concept is therefore developed where exchange of active-site motifs results in a reversible switch between C/O-glycosyltransferase (C/O-GT) activity. Mechanistic proposal for enzymatic C-glycosylation involves a single nucleophilic displacement at the glucosyl anomeric carbon, proceeding through an oxocarbenium ion-like transition state. Alternatively, the reaction could be described as Friedel–Crafts-like direct alkylation of the phenolic acceptor.

LacZ β‐galactosidase: Structure and function of an enzyme of historical and molecular biological importance

This review provides an overview of the structure, function, and catalytic mechanism of lacZ β-galactosidase. The protein played a central role in Jacob and Monod's development of the operon model for the regulation of gene expression. Determination of the crystal structure made it possible to understand why deletion of certain residues toward the amino-terminus not only caused the full enzyme tetramer to dissociate into dimers but also abolished activity. It was also possible to rationalize α-complementation, in which addition to the inactive dimers of peptides containing the "missing" N-terminal residues restored catalytic activity. The enzyme is well known to signal its presence by hydrolyzing X-gal to produce a blue product. That this reaction takes place in crystals of the protein confirms that the X-ray structure represents an active conformation. Individual tetramers of β-galactosidase have been measured to catalyze 38,500 ± 900 reactions per minute. Extensive kinetic, biochemical, mutagenic, and crystallographic analyses have made it possible to develop a presumed mechanism of action. Substrate initially binds near the top of the active site but then moves deeper for reaction. The first catalytic step (called galactosylation) is a nucleophilic displacement by Glu537 to form a covalent bond with galactose. This is initiated by proton donation by Glu461. The second displacement (degalactosylation) by water or an acceptor is initiated by proton abstraction by Glu461. Both of these displacements occur via planar oxocarbenium ion-like transition states. The acceptor reaction with glucose is important for the formation of allolactose, the natural inducer of the lac operon.

Identification of Tyr241 as a Key Catalytic Base in the Family 4 Glycoside Hydrolase BglT from Thermotoga maritima

While the vast majority of glycosidases catalyze glycoside hydrolysis via oxocarbenium ion-like transition states and typically employ carboxylic acid residues as acid/base or nucleophile catalysts, two subfamilies of these enzymes (GH4 and GH109 in the CAZY classification) conduct hydrolysis via a redox-assisted mechanism involving anionic transition states. While good evidence of this mechanism has been obtained, the identities of the catalytic residues involved have not yet been confirmed. Mechanistic analyses of mutants of the 6-phospho-β-glucosidase from Thermotoga maritima (BglT), in which the active site tyrosine residue (Tyr 241) has been replaced with Phe and Ala, provide support for its role as a catalytic base. The pH dependence of k(cat) and k(cat)/K(m), particularly of the acidic limb corresponding to the base, is shifted relative to that of the wild-type enzyme. Kinetic isotope effects for hydrolysis of substrates deuterated at C1, C2, and C3 by the Tyr 241 mutants are strongly pH-dependent, with essentially full primary kinetic isotope effects being observed for the 2-deutero substrate at low pH with the Tyr241Ala mutant. This is consistent with a slowing of the deprotonation step upon removal of the base.

QM/MM study of catalytic mechanism of Xylanase Cex from Cellulomonas fimi

Xylanase Cex from Cellulomonas fimi is a bifunctional enzyme that catalyzes the degradation of both cellulose and xylan. As a result, it might find valuable applications in production of biofuels. In this work, we presented a detailed theoretical investigation of hydrolysis of the xylopentaose molecule catalyzed by Cex, using a hybrid quantum mechanical and molecular mechanical approach. Our results support the experimental observation that the hydrolysis proceeds via the net retention mechanism. More interestingly, our simulations indicate that the xylose unit at −1 binding site should take a boat (B2,5) conformation as a possible reactive conformer, while the oxo-carbenium ion-like transition states take the combination of B2,5/OS2 for glycosylation, and OS2/O,3B for deglycosylation. Our molecular dynamics simulations of mutants further suggest that two catalytic residues (E127 and E233) play the vital role in this ring distortion. Indeed, this conformational change is necessary to facilitate the first step of nucleophilic attack by E233 at the anomeric carbon center.

Essential role of glutamate 317 in galactosyl transfer by α3GalT: a computational study

Retaining glycosyltransferases (ret-GTs) are the enzymes responsible for the biosynthesis of highly specific glycosidic bonds and have drawn the interest of the scientific community. The catalytic mechanism of such enzymes is not yet fully understood and its study remains a challenge for both experimental and theoretical researches. In the case of ret-GTs where a well defined nucleophilic agent is identified in the vicinity of the anomeric center, a double-displacement mechanism via a covalent enzyme-glycosyl intermediate is commonly assumed and has received some experimental support, although not direct and univocal evidence has been obtained so far. This is the case for α-(1→3)-galactosyltransferase (α3GalT), a ret-GT from Bos taurus where a glutamate (Glu317) is in suitable position to act as a nucleophile. Here we perform density functional theory (DFT) quantum mechanics/molecular mechanics (QM/MM) calculations on the full α3GalT enzyme to analyze the role of Glu317 in the catalytic process. This is done not only for the double-displacement mechanism, where the function of the nucleophile is obvious, but also in the scenario of a front-side attack mechanism (via an oxocarbenium ion-like transition state (SNi) or an ion-pair oxocarbenium intermediate (SNi-like)). Glu317 is found to be essential in both cases. For a front-side attack, this residue would have a key role in leaving group departure and consequent stabilization of the increasing positive charge at the anomeric center. This finding alerts on the interpretation of the mutagenesis data as both, the formation of a covalent intermediate and a SNi or a SNi-like mechanism ‘assisted’ by a nucleophile, could be consistent with experiment. In addition, it could explain why the covalent enzyme-glycosyl intermediate has never been isolated.

Retaining Glycosyltransferase Mechanism Studied by QM/MM Methods: Lipopolysaccharyl-α-1,4-galactosyltransferase C Transfers α-Galactose via an Oxocarbenium Ion-like Transition State

Glycosyltransferases (GTs) catalyze the highly specific biosynthesis of glycosidic bonds and, as such, are important both as drug targets and for biotechnological purposes. Despite their broad interest, fundamental questions about their reaction mechanism remain to be answered, especially for those GTs that transfer the sugar with net retention of the configuration at the anomeric carbon (retaining glycosyltransferases, ret-GTs). In the present work, we focus on the reaction catalyzed by lipopolysaccharyl-α-1,4-galactosyltransferase C (LgtC) from Neisseria meningitides. We study and compare the different proposed mechanisms (S(N)i, S(N)i-like, and double displacement mechanism via a covalent glycosyl-enzyme intermediate, CGE) by using density functional theory (DFT) and quantum mechanics/molecular mechanics (QM/MM) calculations on the full enzyme. We characterize a dissociative single-displacement (S(N)i) mechanism consistent with the experimental data, in which the acceptor substrate attacks on the side of the UDP leaving group that acts as a catalytic base. We identify several key interactions that help this front-side attack by stabilizing the transition state. Among them, Gln189, the putative nucleophile in a double displacement mechanism, is shown to favor the charge development at the anomeric center by about 2 kcal/mol, compatible with experimental mutagenesis data. We predict that using 3-deoxylactose as acceptor would result in a reduction of k(cat) to 0.6-3% of that for the unmodified substrates. The reactions of the Q189A and Q189E mutants have also been investigated. For Q189E, there is a change in mechanism since a CGE can be formed which, however, is not able to evolve to products. The current findings are discussed in the light of the available experimental data and compared with those for other ret-GTs.

Catalytic Itinerary in 1,3-1,4-β-Glucanase Unraveled by QM/MM Metadynamics. Charge Is Not Yet Fully Developed at the Oxocarbenium Ion-like Transition State

Retaining glycoside hydrolases (GHs), key enzymes in the metabolism of polysaccharides and glycoconjugates and common biocatalysts used in chemoenzymatic oligosaccharide synthesis, operate via a double-displacement mechanism with the formation of a glycosyl-enzyme intermediate. However, the degree of oxocarbenium ion character of the reaction transition state and the precise conformational itinerary of the substrate during the reaction, pivotal in the design of efficient inhibitors, remain elusive for many GHs. By means of QM/MM metadynamics, we unravel the catalytic itinerary of 1,3-1,4-β-glucanase, one of the most active GHs, belonging to family 16. We show that, in the Michaelis complex, the enzyme environment restricts the conformational motion of the substrate to stabilize a (1,4)B/(1)S(3) conformation of the saccharide ring at the -1 subsite, confirming that this distortion preactivates the substrate for catalysis. The metadynamics simulation of the enzymatic reaction captures the complete conformational itinerary of the substrate during the glycosylation reaction ((1,4)B/(1)S(3) -(4)E/(4)H(3) - (4)C(1)) and shows that the transition state is not the point of maximum charge development at the anomeric carbon. The overall catalytic mechanism is of dissociative type, and proton transfer to the glycosidic oxygen is a late event, clarifying previous kinetic studies of this enzyme.

Mechanistic evidence for a front-side, SNi-type reaction in a retaining glycosyltransferase

A previously determined crystal structure of the ternary complex of trehalose-6-phosphate synthase identified a putative transition state-like arrangement based on validoxylamine A 6'-O-phosphate and uridine diphosphate in the active site. Here linear free energy relationships confirm that these inhibitors are synergistic transition state mimics, supporting front-face nucleophilic attack involving hydrogen bonding between leaving group and nucleophile. Kinetic isotope effects indicate a highly dissociative oxocarbenium ion-like transition state. Leaving group (18)O effects identified isotopically sensitive bond cleavages and support the existence of a hydrogen bond between the nucleophile and departing group. Brønsted analysis of nucleophiles and Taft analysis highlight participation of the nucleophile in the transition state, also consistent with a front-face mechanism. Together, these comprehensive, quantitative data substantiate this unusual enzymatic reaction mechanism. Its discovery should prompt useful reassessment of many biocatalysts and their substrates and inhibitors.

Aromatic interactions at the catalytic subsite of sucrose phosphorylase: Their roles in enzymatic glucosyl transfer probed with Phe 52 → Ala and Phe 52 → Asn mutants

Mutants of Leuconostoc mesenteroides sucrose phosphorylase having active-site Phe 52 replaced by Ala (F52A) or Asn (F52N) were characterized by free energy profile analysis for catalytic glucosyl transfer from sucrose to phosphate. Despite large destabilization (⩾3.5 kcal/mol) of the transition states for enzyme glucosylation and deglucosylation in both mutants as compared to wild-type, the relative stability of the glucosyl enzyme intermediate was weakly affected by substitution of Phe 52. In reverse reaction where fructose becomes glucocylated, “error hydrolysis” was the preponderant path of breakdown of the covalent intermediate of F52A and F52N. It is proposed, therefore, that Phe 52 facilitates reaction of the phosphorylase through (1) positioning of the transferred glucosyl moiety at the catalytic subsite and (2) strong cation-π stabilization of the oxocarbenium ion-like transition states flanking the covalent enzyme intermediate.

Transition State of ADP-Ribosylation of Acetyllysine Catalyzed by Archaeoglobus fulgidus Sir2 Determined by Kinetic Isotope Effects and Computational Approaches

Sirtuins are protein-modifying enzymes distributed throughout all forms of life. These enzymes bind NAD(+), a universal metabolite, and react it with acetyllysine residues to effect deacetylation of protein side chains. This NAD(+)-dependent deacetylation reaction has been observed for sirtuin enzymes derived from archaeal, eubacterial, yeast, metazoan, and mammalian species, suggesting conserved chemical mechanisms for these enzymes. The first chemical step of deacetylation is the reaction of NAD(+) with an acetyllysine residue which forms an enzyme-bound ADPR-peptidylimidate intermediate and nicotinamide. In this manuscript, the transition state for the ADP-ribosylation of acetyllysine is solved for an Archaeoglobus fulgidus sirtuin (Af2Sir2). Kinetic isotope effects (KIEs) were obtained by the competitive substrate method and were [1(N)-(15)N] = 1.024(2), [1'(N)-(14)C] = 1.014(4), [1'(N)-(3)H] = 1.300(3), [2'(N)-(3)H] = 1.099(5), [4'(N)-(3)H] = 0.997(2), [5'(N)-(3)H] = 1.020(5), [4'(N)-(18)O] = 0.984(5). KIEs were calculated for candidate transition state structures using computational methods (Gaussian 03 and ISOEFF 98) in order to match computed and experimentally determined KIEs to solve the transition state. The results indicate that the enzyme stabilizes a highly dissociated oxocarbenium ionlike transition state with very low bond orders to the leaving group nicotinamide and the nucleophile acetyllysine. A concerted yet highly asynchronous substitution mechanism forms the ADPR-peptidylimidate intermediate of the sirtuin deacetylation reaction.

Glycosyltransferase Mechanisms: Impact of a 5-Fluoro Substituent in Acceptor and Donor Substrates on Catalysis

In glycosyltransferase-catalyzed reactions a new carbohydrate-carbohydrate bond is formed between a carbohydrate acceptor and the carbohydrate moiety of either a sugar nucleotide donor or lipid-linked saccharide donor. It is currently believed that most glycosyltransferase-catalyzed reactions occur via an electrophilic activation mechanism with the formation of an oxocarbenium ion-like transition state, a hypothesis that makes clear predictions regarding the charge development on the donor (strong positive charge) and acceptor (minimal negative charge) substrates. To better understand the mechanism of these enzyme-catalyzed reactions, we have introduced a strongly electron-withdrawing group (fluorine) at C-5 of both donor and acceptor substrates in order to explore its effect on catalysis. In particular, we have investigated the effects of the 5-fluoro analogues on the kinetics of two glycosyltransferase-catalyzed reactions mediated by UDP-GlcNAc:GlcNAc-P-P-Dol N-acetylglucosaminyltransferase (chitobiosyl-P-P-lipid synthase, CLS) and beta-N-acetylglucosaminyl-beta-1,4 galactosyltransferase (GalT). The 5-fluoro group has a marked effect on catalysis when inserted into the UDP-GlcNAc donor, with the UDP(5-F)-GlcNAc serving as a competitive inhibitor of CLS rather than a substrate. The (5-F)-GlcNAc beta-octyl glycoside acceptor, however, is an excellent substrate for GalT. Both of these results support a weakly associative transition state for glycosyltransferase-catalyzed reactions that proceed with inversion of configuration.

Probing the active site of Corynebacterium callunae starch phosphorylase through the characterization of wild-type and His334→Gly mutant enzymes

His334 facilitates catalysis by Corynebacterium callunae starch phosphorylase through selective stabilization of the transition state of the reaction, partly derived from a hydrogen bond between its side chain and the C-6 hydroxy group of the glucosyl residue undergoing transfer to and from phosphate. We have substituted His334 by a Gly and measured the disruptive effects of the site-directed replacement on active site function using steady-state kinetics and NMR spectroscopic characterization of the cofactor pyridoxal 5'-phosphate and binding of carbohydrate ligands. Purified H334G showed 0.05% and 1.3% of wild-type catalytic center activity for phosphorolysis of maltopentaose (kcatP = 0.033 s(-1)) and substrate binding affinity in the ternary complex with enzyme bound to phosphate (Km = 280 mm), respectively. The 31P chemical shift of pyridoxal 5'-phosphate in the wild-type was pH-dependent and not perturbed by binding of arsenate. At pH 7.25, it was not sensitive to the replacement His334-->Gly. Analysis of interactions of alpha-d-glucose 1-phosphate and alpha-d-xylose 1-phosphate upon binding to wild-type and H334G phosphorylase, derived from saturation transfer difference NMR experiments, suggested that disruption of enzyme-substrate interactions in H334G was strictly local, affecting the protein environment of sugar carbon 6. pH profiles of the phosphorolysis rate for wild-type and H334G were both bell-shaped, with the broad pH range of optimum activity in the wild-type (pH 6.5-7.5) being narrowed and markedly shifted to lower pH values in the mutant (pH 6.5-7.0). External imidazole partly restored the activity lost in the mutant, without, however, participating as an alternative nucleophile in the reaction. It caused displacement of the entire pH profile of H334G by + 0.5 pH units. A possible role for His334 in the formation of the oxocarbenium ion-like transition state is suggested, where the hydrogen bond between its side chain and the 6-hydroxyl polarizes and positions O-6 such that electron density in the reactive center is enhanced.

Crystal Structures of Pasteurella multocida Sialyltransferase Complexes with Acceptor and Donor Analogues Reveal Substrate Binding Sites and Catalytic Mechanism,

Sialyltransferases are key enzymes involved in the biosynthesis of biologically and pathologically important sialic acid-containing molecules in nature. Binary X-ray crystal structures of a multifunctional Pasteurella multocida sialyltransferase (Delta24PmST1) with a donor analogue CMP-3F(a)Neu5Ac or CMP-3F(e)Neu5Ac were determined at 2.0 and 1.9 A resolutions, respectively. Ternary X-ray structures of the protein in complex with CMP or a donor analogue CMP-3F(a)Neu5Ac and an acceptor lactose have been determined at 2.0 and 2.27 A resolutions, respectively. This represents the first sialyltransferase structure and the first GT-B-type glycosyltransferase structure that is bound to both a donor analogue and an acceptor simultaneously. The four structures presented here reveal that binding of the nucleotide-activated donor sugar causes a buried tryptophan to flip out of the protein core to interact with the donor sugar and helps define the acceptor sugar binding site. Additionally, key amino acid residues involved in the catalysis have been identified. Structural and kinetic data support a direct displacement mechanism involving an oxocarbenium ion-like transition state assisted with Asp141 serving as a general base to activate the acceptor hydroxyl group.

Structural Basis for the Mechanistic Understanding of Human CD38-controlled Multiple Catalysis

The enzymatic cleavage of the nicotinamide-glycosidic bond on nicotinamide adenine dinucleotide (NAD(+)) has been proposed to go through an oxocarbenium ion-like transition state. Because of the instability of the ionic intermediate, there has been no structural report on such a transient reactive species. Human CD38 is an ectoenzyme that can use NAD(+) to synthesize two calcium-mobilizing molecules. By using NAD(+) and a surrogate substrate, NGD(+), we captured and determined crystal structures of the enzyme complexed with an intermediate, a substrate, and a product along the reaction pathway. Our results showed that the intermediate is stabilized by polar interactions with the catalytic residue Glu(226) rather than by a covalent linkage. The polar interactions between Glu(226) and the substrate 2',3'-OH groups are essential for initiating catalysis. Ser(193) was demonstrated to have a regulative role during catalysis and is likely to be involved in intermediate stabilization. In addition, a product inhibition effect by ADP-ribose (through the reorientation of the product) or GDP-ribose (through the formation of a covalently linked GDP-ribose dimer) was observed. These structural data provide insights into the understanding of multiple catalysis and clues for drug design.