Exogenous α-amylase improves the digestibility of corn and corn-soybean meal diets for broilers.

Starch is the main energy source in broiler diets. However, endogenous amylase secretion in young broilers is suboptimal to completely digest dietary starch, so exogenous α-amylase supplementation may help increase starch digestibility. The objective of this study was to assess the supplementation of increasing doses of an exogenous α-amylase (0, 40, 80, 120, and 160 kilo-novo α-amylase units (KNU)/kg) on corn and on a complete corn-soybean meal diet for 25-day-old broilers. Jejunal and ileal apparent digestibility coefficients of available starch, resistant starch, total starch, and DM, DM total tract retention, as well as dietary AME levels were evaluated. Interactions (P < 0.05) between diets and α-amylase showed that the enzyme had a more evident effect on increasing DM jejunal digestibility and AME on corn compared with the complete diet. Corn DM digestibility increased to a maximum of 67.84% with up to 47 KNU/kg, whereas 89 KNU/kg led to a maximum of 53.92% in the complete diet A maximum increase of 64 kcal AME/kg was obtained with 80 KNU/kg on the complete diet, whereas 109 KNU/kg generated 327 kcal AME/kg on corn (P < 0.05). Increasing the α-amylase dose linearly increased ileal digestibility of resistant starch (P < 0.05), and the effect on DM total tract retention was quadratic (P < 0.05). Corn showed a higher digestibility for DM, resistant and total starch, as well as DM total tract retention and AME, compared with the complete diet (P < 0.05). Treatments had no influence on available starch. The inclusion of exogenous α-amylase improves starch, DM, and energy utilization of corn-based and corn-soybean meal-based diets for broilers.

Effects of feed form and energy levels on growth performance, carcass yield and nutrient digestibility in broilers.

Feed form is well recognized to improve broiler performance, specially by increasing feed intake (FI). However, when different diet energy levels are used, the results differ in the literature. Therefore, this experiment was conducted to evaluate the influence of feed form and dietary metabolizable energy (ME) levels on broiler performance, carcass yield and on the digestibility of DM, CP, starch and gross energy. In total, 1152 male Cobb 500 broilers were evaluated between 35 and 47 days. The birds were distributed according to a completely randomized design in a 2 × 4 factorial arrangement, consisting of two feed forms (mash or pellet) and four ME levels (12.73, 13.06, 13.40 or 13.73 MJ/kg), totaling eight treatments with eight replicates of 18 birds. Broilers fed the lowest ME level presented the lowest weight gain (WG) and worst feed per unit gain (P < 0.01). Metabolizable energy intake increased (P < 0.01) with progressive increments of ME, which, however, did not affect caloric conversion (CC, P > 0.05). Pelleted diets promoted higher FI, WG, ME intake (P < 0.01) and better feed per unit gain and CC (P < 0.05) compared with mash. In mash diets, increasing dietary ME levels promoted a linear increase in WG (P < 0.01) and reduced feed per unit gain (P ≤ 0.05), but did not affect FI (P > 0.05). In pelleted diets, on the other hand, increasing ME levels linearly reduced FI (P < 0.05) and feed per unit gain (P < 0.01). Broilers fed pelleted diets presented higher abdominal fat deposition than those fed mash (P < 0.05). Increasing ME levels reduced the coefficients of ileal apparent digestibility of DM (P < 0.01) and total starch (P < 0.05) but did not affect the digestibility of other evaluated nutrients. The digestibility of all nutrients was lower when pelleted diets were fed compared with mash. Increasing inert material inclusion in the diets at the expense of soybean oil to reduce dietary ME levels promoted higher pellet durability index values (P < 0.05) and the percentage of fines (P < 0.01). Overall, the results suggest that pelleted diets promote better broiler performance because they increase FI, since the digestibility of dietary fractions is reduced. Chickens consuming low-energy pelleted diets may increase FI to compensate for energy deficit. In contrast, broilers fed mash diets may have reached their maximum intake capacity and did not regulate FI by changing feed energy density. When feeding pelleted diets, dietary energy reduction should be considered to reduce feed costs and to improve the carcass quality of broilers.

Interaction between xylanase and phytase on the digestibility of corn and a corn/soy diet for broiler chickens.

An experiment was carried out to evaluate the digestibility and metabolizability of corn and a corn/soy-based diet with the inclusion or not of xylanase and/or phytase in broilers. In the trial, 1,120 broiler chicks were distributed according to a completely randomized experimental design, consisting of 16 treatments, with 10 replicates of 7 birds each. Treatments were evaluated following a factorial arrangement (4 × 2 × 2), with 4 xylanase levels (zero, 50, 100, or 150 fungal ß-xylanase units/kg; FXU), 2 phytase levels (zero or 1,000 phytase units/kg; FTU), and 2 diets (corn/soy or pure corn). The same basal diets were fed from one to 14 d post hatch for all birds, after which the experimental diets were provided until d 25. All birds were euthanized on d 25 for collection of ileal contents. Samples of feed, excreta, and ileal digesta were analyzed for determination of apparent ileal digestibility. The effect of xylanase on the coefficient of apparent dry matter metabolizability and apparent metabolizable energy was increased by the presence of phytase in the complete diet but not in the diet based on pure corn resulting in a diet*phytase*xylanase interaction (P < 0.01; P < 0.001, respectivaly). Equivalent effects were observed for the apparent coefficient of ileal protein digestibility in which xylanase effects were potentiated by the presence of phytase only in the complete diet, resulting in a significant 3-way interaction. In corn there was a limitation in improving digestibility when we added increasing levels of xylanase with phytase. Otherwise in the corn/soy-based diets, the enzymes were potencialized when they were added together. The fact that the effect of xylanase was enhanced by the presence of phytase in complete diets but not in pure corn may be associated with differences in substrate (arabinoxylan and/or phytate) concentration and presentation, diet nutrient balance, or other factors. It can be concluded that the interactive effects of xylanase and phytase can be substantial but may depend on the characteristics of the diet fed.

Effects of different limestone particle sizes in the diet of broiler breeders post molting on their performance, egg quality, incubation results, and pre-starter performance of their progeny.

An experiment was conducted to test the hypothesis that a coarse limestone diet improves productivity, reproductive performance and the calcium utilization of molted broiler breeders. In total, 640 broiler breeder females, 73-week-old and sixty-four 27-week-old cockerels, Cobb 500, were evaluated during 10 weeks, according to a randomized block design composed of 4 treatments with 8 replicates each. Treatments consisted of diets with the inclusion of 100% fine limestone-fine PS (0.2 mm GMD-geometric mean diameter); PS1: 30% fine limestone+70% limestone with 1.0 mm GMD; PS2: 30% fine limestone+70% limestone with 2.0 mm GMD; and PS3: 30% fine limestone+70% limestone with 3.0 mm GMD. Calcium retention in the gizzard of the breeders, bone characteristics, and breeder performance, egg characteristics, eggshell quality, incubation performance, chick quality and yield, chick pre-starter live performance, and chick bone characteristics were determined. There was no significant difference (P>0.05) in the rate of lay, percentage of non-settable eggs, egg weight, egg shape index, egg specific gravity, eggshell weight, thickness, and percentage hatchability and egg weight loss of broiler breeders fed with diets with different limestone particle sizes. The chick quality and yield, chick pre-starter live performance, and chick bone characteristics were not affected (P>0.05) by any of the limestone particle sizes. It was concluded that live and reproductive performance parameters of broiler breeders post molting is not affected by limestone particle size in the feed.

Chemistry of gene silencing: the mechanism of NAD+-dependent deacetylation reactions.

The Sir2 enzyme family is responsible for a newly classified chemical reaction, NAD(+)-dependent protein deacetylation. New peptide substrates, the reaction mechanism, and the products of the acetyl transfer to NAD(+) are described for SIR2. The final products of SIR2 reactions are the deacetylated peptide and the 2' and 3' regioisomers of O-acetyl ADP ribose (AADPR), formed through an alpha-1'-acetyl ADP ribose intermediate and intramolecular transesterification reactions (2' --> 3'). The regioisomers, their anomeric forms, the interconversion rates, and the reaction equilibria were characterized by NMR, HPLC, 18O exchange, and MS methods. The mechanism of acetyl transfer to NAD(+) includes (1) ADP ribosylation of the peptide acyl oxygen to form a high-energy O-alkyl amidate intermediate, (2) attack of the 2'-OH group on the amidate to form a 1',2'-acyloxonium species, (3) hydrolysis to 2'-AADPR by the attack of water on the carbonyl carbon, and (4) an SIR2-independent transesterification equilibrating the 2'- and 3'-AADPRs. This mechanism is unprecedented in ADP-ribosyl transferase enzymology. The 2'- and 3'-AADPR products are candidate molecules for SIR2-initiated signaling pathways.

Atomic motion in enzymatic reaction coordinates.

Atomic excursions of reactants in enzymatic catalytic sites can be estimated from high-resolution crystal structures of enzyme complexes with substrates, transition state analog inhibitors and products. Transition state structures, defined from kinetic isotope effect studies, are compared to crystallographic structures to validate the properties of the transition state analog. Atomic excursions in enzymatic catalytic sites can differ from those in solution and define the role of the enzymatic catalyst in directing atomic motion.

Transition state variation in enzymatic reactions.

Experimental analysis of enzymatic transition states by kinetic isotope effect methods has established geometric variation in related transition state structures. Differences are apparent in development of the reaction coordinate, in solvolytic transition states relative to those in enzymatic catalytic sites, in the stereochemistry of related substrates at the transition state, and in reactions catalyzed by related enzymes.

Management of nasopharyngeal salivary gland malignancy.

OBJECTIVE: The objective of this study was to evaluate the oncological outcome and complication rate following surgical treatment of nasopharyngeal salivary gland malignancy. STUDY DESIGN: Retrospective case review at tertiary care skull base center. METHODS: Pertinent medical records were reviewed from 23 patients presenting with minor salivary gland malignancy. Clinical presentation, prior treatment, histological type and grade, clinical stage, details of surgical treatment, and postoperative adjuvant radiation therapy were studied. Survival and recurrence data were analyzed using the Kaplan-Meier and Cox proportional hazards methods. RESULTS: Histological types included 11 adenoid cystic carcinomas, 8 mucoepidermoid carcinomas, and 4 cases of adenocarcinoma not otherwise specified. All patients underwent primary surgical resection, and the lateral infratemporal middle fossa approach was used in 20 patients. Prior radiation therapy had been administered in 6 patients who presented for treatment of recurrent disease, and the remaining 17 patients underwent planned postoperative radiation therapy. Elective neck dissection was undertaken in 15 patients, and occult neck disease was present in 47%. Disease specific survival was 67% at 5 years and 48% at 10 years. High-grade tumors had a significantly poorer outcome (P =.035) with a relative risk of 4.6 compared with low-grade disease. Local control was seen to be 77% at 5 years. CONCLUSIONS: Planned combined surgery and radiation therapy achieves survival outcomes and recurrence rates in nasopharyngeal salivary gland malignancy comparable to results reported using the same treatment for minor salivary gland tumors cancer originating elsewhere in the head and neck. Because of the high rate of occult neck metastases, we recommend elective neck dissection as part of the surgical treatment with this disease entity. The lateral infratemporal middle fossa approach provides safe and adequate access to resect the vast majority of these tumors with acceptable complication rates. A reliable form of vascularized reconstruction is necessary to prevent serious postoperative complications, and we currently prefer the gastro-omental free flap.

Structural analysis of adenine phosphoribosyltransferase from Saccharomyces cerevisiae.

Adenine phosphoribosyltransferase (APRTase) is a widely distributed enzyme, and its deficiency in humans causes the accumulation of 2,8-dihydroxyadenine. It is the sole catalyst for adenine recycling in most eukaryotes. The most commonly expressed APRTase has subunits of approximately 187 amino acids, but the only crystal structure is from Leishmania donovani, which expresses a long form of the enzyme with 237 residues. Saccharomyces cerevisiae APRTase was selected as a representative of the short APRTases, and the structure of the apo-enzyme and sulfate bound forms were solved to 1.5 and 1.75 A, respectively. Yeast APRTase is a dimeric molecule, and each subunit is composed of a central five-stranded beta-sheet surrounded by five alpha-helices, a structural theme found in all known purine phosphoribosyltransferases. The structures reveal several important features of APRTase function: (i) sulfate ions bound at the 5'-phosphate and pyrophosphate binding sites; (ii) a nonproline cis peptide bond (Glu67-Ser68) at the pyrophosphate binding site in both apo-enzyme and sulfate-bound forms; and (iii) a catalytic loop that is open and ordered in the apo-enzyme but open and disordered in the sulfate-bound form. Alignment of conserved amino acids in short-APRTases from 33 species reveals 13 invariant and 15 highly conserved residues present in hinges, catalytic site loops, and the catalytic pocket. Mutagenesis of conserved residues in the catalytic loop, subunit interface, and phosphoribosylpyrophosphate binding site indicates critical roles for the tip of the catalytic loop (Glu106) and a catalytic site residue Arg69, respectively. Mutation of one loop residue (Tyr103Phe) increases k(cat) by 4-fold, implicating altered dynamics for the catalytic site loop.

Addition of lithiated 9-deazapurine derivatives to a carbohydrate cyclic imine: convergent synthesis of the aza-C-nucleoside immucillins.

Means have been developed for the synthesis and addition of 9-deaza-9-lithiopurine derivatives to the carbohydrate-derived cyclic imine 6 in facile convergent syntheses of biologically active aza-C-nucleosides.

A transition-state analogue reduces protein dynamics in hypoxanthine-guanine phosphoribosyltransferase.

Hypoxanthine-guanine phosphoribosyltransferase (HGPRT) is the key enzyme in purine base salvage in humans and in purine auxotrophs, including Plasmodium falciparum, the leading cause of malaria. Hydrogen/deuterium (H/D) exchange into amide bonds, quantitated by on-line HPLC and mass spectrometry, has been used to compare the dynamic and conformational properties of human HGPRT alone, the HGPRT-GMP-Mg(2+) complex, the HGPRT-IMP-MgPPi <==> HGPRT-Hx-MgPRPP equilibrating mixture, and the transition-state analogue complex HGPRT-ImmGP-MgPPi. The rate and extent of H/D exchange of 26 peptic peptides, spanning 91% of the primary structure, have been monitored. Human HGPRT has 207 amide H/D exchange sites. After 1 h in D2O, HGPRT alone exchanges 160, HGPRT-GMP-Mg(2+) exchanges 154, the equilibrium complex exchanges 139, and the transition-state analogue complex exchanges 126 of these amide protons. H/D exchange rates are correlated with structure for peptides in (1) catalytic site loops, (2) a connected peptide of the subunit interface of the tetramer, and (3) a loop buried in the catalytic site. Structural properties related to H/D exchange are defined from crystallographic studies of the HGPRT-GMP-Mg(2+) and HGPRT-ImmGP-MgPPi complexes. Transition-state analogue binding strengthens the interaction between subunits and tightens the catalytic site loops. The solvent exchange dynamics in specific peptides correlates with hydrogen bond patterns, solvent access, crystallographic B-factors, and ligand exchange rates. Solvent exchange reveals loop dynamics in the free enzyme, Michaelis complexes, and the complex with the bound transition-state analogue. Proton transfer paths, rather than dynamic motion, are required to explain exchange into a buried catalytic site peptide in the complex with the bound transition-state analogue.

Purine nucleoside phosphorylase from Mycobacterium tuberculosis. Analysis of inhibition by a transition-state analogue and dissection by parts.

Purine salvage pathways are predicted to be present from the genome sequence of Mycobacterium tuberculosis. The M. tuberculosis deoD gene encodes a presumptive purine nucleoside phosphorylase (PNP). The gene was cloned, expressed, purified, and found to exhibit PNP activity. Purified M. tuberculosis PNP is trimeric, similar to mammalian PNP's but unlike the hexameric Escherichia coli enzyme. Immucillin-H is a rationally designed analogue of the transition state that has been shown to be a potent inhibitor of mammalian PNP's. This inhibitor also exhibits slow-onset inhibition of M. tuberculosis PNP with a rapid, reversible inhibitor binding (K(i) of 2.2 nM) followed by an overall dissociation constant (K(i)) of 28 pM, yielding a K(m)/K(i) value of 10(6). Time-dependent tight binding of the inhibitor occurs with a rate of 0.1 s(-)(1), while relaxation of the complex is slower at 1.4 x 10(-)(3) s(-)(1). The pH dependence of the K(i) value of immucillin-H to the M. tuberculosis PNP suggests that the inhibitor binds as the neutral, unprotonated form that is subsequently protonated to generate the tight-binding species. The M. tuberculosis enzyme demonstrates independent and equivalent binding of immucilin-H at each of the three catalytic sites, unlike mammalian PNP. Analysis of the components of immucillin-H confirms that the inhibition gains most of its binding energy from the 9-deazahypoxanthine group (K(is) of 0.39 microM) while the 1,4-dideoxy-1,4-iminoribitol binds weakly (K(is) of 2.9 mM). Double-inhibition studies demonstrate antagonistic binding of 9-deazahypoxanthine and iminoribitol (beta = 13). However, the covalent attachment of these two components in immucillin-H increases equilibrium binding affinity by a factor of >14 000 (28 pM vs 0.39 microM) compared to 9-deazahypoxanthine alone, and by a factor of >10(8) compared to iminoribitol alone (28 pM vs 2.9 mM), from initial velocity measurements. The structural basis for M. tuberculosis PNP inhibition by immucillin-H and by its component parts is reported in the following paper [Shi, W., Basso, L. A., Santos, D. S., Tyler, P. C., Furneaux, R. H., Blanchard, J. S., Almo, S. C., and Schramm, V. L. (2001) Biochemistry 40, 8204-8215].

Structures of purine nucleoside phosphorylase from Mycobacterium tuberculosis in complexes with immucillin-H and its pieces.

A structural genomics comparison of purine nucleoside phosphorylases (PNPs) indicated that the enzyme encoded by Mycobacterium tuberculosis (TB-PNP) resembles the mammalian trimeric structure rather than the bacterial hexameric PNPs. The crystal structure of M. tuberculosis PNP in complex with the transition-state analogue immucillin-H (ImmH) and inorganic phosphate was solved at 1.75 A resolution and confirms the trimeric structure. Binding of the inhibitor occurs independently at the three catalytic sites, unlike mammalian PNPs which demonstrate negative cooperativity in ImmH binding. Reduced subunit interface contacts for TB-PNP, compared to the mammalian enzymes, correlate with the loss of the cooperative inhibitor binding. Mammalian and TB-PNPs both exhibit slow-onset inhibition and picomolar dissociation constants for ImmH. The structure supports a catalytic mechanism of reactant destabilization by neighboring group electrostatic interactions, transition-state stabilization, and leaving group activation. Despite an overall amino acid sequence identity of 33% between bovine and TB-PNPs and almost complete conservation in active site residues, one catalytic site difference suggests a strategy for the design of transition-state analogues with specificity for TB-PNP. The structure of TB-PNP was also solved to 2.0 A with 9-deazahypoxanthine (9dHX), iminoribitol (IR), and PO(4) to reconstruct the ImmH complex with its separate components. One subunit of the trimer has 9dHX, IR, and PO(4) bound, while the remaining two subunits contain only 9dHX. In the filled subunit, 9dHX retains the contacts found in the ImmH complex. However, the region of IR that corresponds to the oxocarbenium ion is translocated in the direction of the reaction coordinate, and the nucleophilic phosphate rotates away from the IR group. Loose packing of the pieces of ImmH in the catalytic site establishes that covalent connectivity in ImmH is required to achieve the tightly bound complex.

Conformational equilibrium isotope effects in glucose by (13)C NMR spectroscopy and computational studies.

Anomeric equilibrium isotope effects for dissolved sugars are required preludes to understanding isotope effects for these molecules bound to enzymes. This paper presents a full molecule study of the alpha- and beta-anomeric forms of D-glucopyranose in water using deuterium conformational equilibrium isotope effects (CEIE). Using 1D (13)C NMR, we have found deuterium isotope effects of 1.043 +/- 0.004, 1.027 +/- 0.005, 1.027 +/- 0.004, 1.001 +/- 0.003, 1.036 +/- 0.004, and 0.998 +/- 0.004 on the equilibrium constant, (H/D)K(beta/alpha), in [1-(2)H]-, [2-(2)H]-, [3-(2)H]-, [4-(2)H]-, [5-(2)H]-, and [6,6'-(2)H(2)]-labeled sugars, respectively. A computational study of the anomeric equilibrium in glucose using semiempirical and ab initio methods yields values that correlate well with experiment. Natural bond orbital (NBO) analysis of glucose and dihedral rotational equilibrium isotope effects in 2-propanol strongly imply a hyperconjugative mechanism for the isotope effects at H1 and H2. We conclude that the isotope effect at H1 is due to n(p) --> sigma* hyperconjugative transfer from O5 to the axial C1--H1 bond in beta-glucose, while this transfer makes no contribution to the isotope effect at H5. The isotope effect at H2 is due to rotational restriction of OH2 at 160 degrees in the alpha form and 60 degrees in the beta-sugar, with concomitant differences in n --> sigma* hyperconjugative transfer from O2 to CH2. The isotope effects on H3 and H5 result primarily from syn-diaxial steric repulsion between these and the axial anomeric hydroxyl oxygen in alpha-glucose. Therefore, intramolecular effects play an important role in isotopic perturbation of the anomeric equilibrium. The possible role of intermolecular effects is discussed in the context of recent molecular dynamics studies on aqueous glucose.

Ricin A-chain inhibitors resembling the oxacarbenium ion transition state.

Ricin toxin A-chain (RTA) is expressed by the castor bean plant and is among the most potent mammalian toxins. Upon activation in the cytosol, RTA depurinates a single adenine from position 4324 of rat 28S ribosomal RNA, causing inactivation of ribosomes by preventing the binding of elongation factors. Kinetic isotope effect studies have established that RTA operates via a D(N)*A(N) mechanism involving an oxacarbenium ion intermediate with bound adenine [Chen, X.-Y., Berti, P. J., and Schramm, V. L. (2000) J. Am. Chem. Soc. 122, 1609-1617]. On the basis of this information, stem-loop RNA molecules were chemically synthesized, incorporating structural features of the oxacarbenium ion-like transition state. A 10-base RNA stem-loop incorporating (1S)-1-(9-deazaadenin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds four times better (0.57 microM) than the 10-base RNA stem-loop with adenosine at the depurination site (2.2 microM). A 10-base RNA stem-loop with 1,2-dideoxyribitol [(2R,3S)-2-(hydroxymethyl)-3-hydroxytetrahydrofuran] at the depurination site binds with a Kd of 3.2 microM and tightens to 0.75 microM in the presence of 9-deazaadenine. A similar RNA stem-loop with 1,4-dideoxy-1,4-imino-D-ribitol at the depurination site binds with a K(d) of 1.3 microM and improves to 0.65 micro;M with 9-deazaadenine added. When (3S,4R)-4-hydroxy-3-(hydroxymethyl)pyrrolidine was incorporated at the depurination site of a 14-base RNA stem-loop, the Kd was 0.48 microM. Addition of 9-deazaadenine tightens the binding to 0.10 microM whereas added adenine increases the affinity to 12 nM. The results of this study are consistent with the unusual dissociative D(N)*A(N) mechanism determined for RTA. Knowledge of this intermediate has led to the design and synthesis of the highest affinity inhibitor reported for the catalytic site of RTA.

Immucillin H, a powerful transition-state analog inhibitor of purine nucleoside phosphorylase, selectively inhibits human T lymphocytes.

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

Transition state structure of purine nucleoside phosphorylase and principles of atomic motion in enzymatic catalysis.

Immucillin-H [ImmH; (1S)-1-(9-deazahypoxanthin-9-yl)-1,4-dideoxy-1,4-imino-D-ribitol] is a 23 pM inhibitor of bovine purine nucleoside phosphorylase (PNP) specifically designed as a transition state mimic [Miles, R. W., Tyler, P. C., Furneaux, R. H., Bagdassarian, C. K., and Schramm, V. L. (1998) Biochemistry 37, 8615-8621]. Cocrystals of PNP and the inhibitor are used to provide structural information for each step through the reaction coordinate of PNP. The X-ray crystal structure of free ImmH was solved at 0.9 A resolution, and a complex of PNP.ImmH.PO(4) was solved at 1.5 A resolution. These structures are compared to previously reported complexes of PNP with substrate and product analogues in the catalytic sites and with the experimentally determined transition state structure. Upon binding, ImmH is distorted to a conformation favoring ribosyl oxocarbenium ion formation. Ribosyl destabilization and transition state stabilization of the ribosyl oxocarbenium ion occur from neighboring group interactions with the phosphate anion and the 5'-hydroxyl of the ribosyl group. Leaving group activation of hypoxanthine involves hydrogen bonds to O6, N1, and N7 of the purine ring. Ordered water molecules provide a proton transfer bridge to O6 and N7 and permit reversible formation of these hydrogen bonds. Contacts between PNP and catalytic site ligands are shorter in the transition state analogue complex of PNP.ImmH.PO(4) than in the Michaelis complexes of PNP.inosine.SO(4) or PNP.hypoxanthine.ribose 1-PO(4). Reaction coordinate motion is dominated by translation of the carbon 1' of ribose between relatively fixed phosphate and purine groups. Purine and pyrimidine phosphoribosyltransferases and nucleoside N-ribosyl hydrolases appear to operate by a similar mechanism.

5'-Deoxyadenosine contacts the substrate radical intermediate in the active site of ethanolamine ammonia-lyase: 2H and 13C electron nuclear double resonance studies.

The mechanism of propagation of the radical center between the cofactor, substrate, and product in the adenosylcobalamin- (AdoCbl) dependent reaction of ethanolamine ammonia-lyase has been probed by pulsed electron nuclear double resonance (ENDOR) spectroscopy. The radical of S-2-aminopropanol, which appears in the steady state of the reaction, was used in ENDOR experiments to determine the nuclear spin transition frequencies of (2)H introduced from either deuterated substrate or deuterated coenzyme and of (13)C introduced into the ribosyl moiety of AdoCbl. A (2)H doublet (1.4 MHz splitting) was observed centered about the Larmor frequency of (2)H. Identical ENDOR frequencies were observed for (2)H irrespective of its mode of introduction into the complex. A (13)C doublet ENDOR signal was observed from samples prepared with [U-(13)C-ribosyl]-AdoCbl. The (13)C coupling tensor obtained from the ENDOR powder pattern shows that the (13)C has scalar as well as dipole-dipole coupling to the unpaired electron located at C1 of S-2-aminopropanol. The dipole-dipole coupling is consistent with a distance of 3.4+/-0.2 A between C1 of the radical and C5' of the labeled cofactor component. These results establish that the C5' carbon of the 5'-deoxyadenosyl radical moves approximately 7 A from its position as part of AdoCbl to a position where it is in contact with C1 of the substrate which lies approximately 12 A from the Co(2+) of cob(II)alamin. These findings are also consistent with the contention that 5'-deoxyadenosine is the sole mediator of hydrogen transfers in ethanolamine ammonia-lyase.

Immucillin-H binding to purine nucleoside phosphorylase reduces dynamic solvent exchange.

The rate and extent of hydrogen/deuterium (H/D) exchange into purine nucleoside phosphorylase (PNP) was monitored by electrospray ionization mass spectrometry (ESI-MS) to probe protein conformational and dynamic changes induced by a substrate analogue, products, and a transition state analogue. The genetic deficiency of PNP in humans is associated with severe T-cell immunodeficiency, while B-cell immunity remains functional. Inhibitors of PNP have been proposed for treatment of T-cell leukemia, to suppress the graft-vs.-host response, or to counter type IV autoimmune diseases without destroying humoral immunity. Calf spleen PNP is a homotrimer of polypeptide chains with 284 amino residues, molecular weight 31,541. Immucillin-H inhibits PNP with a Kd of 23 pM when only one of the three catalytic sites is occupied. Deuterium exchange occurs at 167 slow-exchange sites in 2 h when no catalytic site ligands are present. The substrate analogue and product prevented H/D exchange at 10 of the sites. Immucillin-H protected 32 protons from exchange at full saturation. When one of the three subunits of the homotrimer is filled with immucillin-H, and 27 protons are protected from exchange in all three subunits. Deuterium incorporation in peptides from residues 132-152 decreased in all complexes of PNP. The rate and/or extent of deuterium incorporation in peptides from residues 29-49, 50-70, 81-98, and 112-124 decreased only in the complex with the transition state analogue. The peptide-specific H/D exchange demonstrates that (1) the enzyme is most compact in the complex with immucillin-H, and (2) filling a single catalytic site of the trimer reduces H/D exchange in the same peptides in adjacent subunits. The peptides most highly influenced by the inhibitor surround the catalytic site, providing evidence for reduced protein dynamic motion caused by the transition state analogue.

Crystal structures of Giardia lamblia guanine phosphoribosyltransferase at 1.75 A(,).

Giardia lamblia, the protozoan parasite responsible for giardiasis, requires purine salvage from its host for RNA and DNA synthesis. G. lamblia expresses an unusual purine phosphoribosyltransferase with a high specificity for guanine (GPRTase). The enzyme's sequence significantly diverges from those of related enzymes in other organisms. The transition state analogue immucillinGP is a powerful inhibitor of HGXPRTase from malaria [Li, C. M., et al. (1999) Nat. Struct. Biol. 6, 582-587] and is also a 10 nM inhibitor of G. lamblia GPRTase. Cocrystallization of GPRTase with immucillinGP led unexpectedly to a GPRTase.immucillinG binary complex with an open catalytic site loop. Diffusion of ligands into preformed crystals gave a GPRTase.immucillinGP.Mg(2+).pyrophosphate complex in which the open loop is stabilized by crystal contacts. G. lamblia GPRTase exhibits substantial structural differences from known purine phosphoribosyltransferases at positions remote from the catalytic site, but conserves most contacts to the bound inhibitor. The filled catalytic site with an open catalytic loop provides insight into ligand binding. One active site Mg(2+) ion is chelated to pyrophosphate, but the other is chelated to two conserved catalytic site carboxylates, suggesting a role for these amino acids. This arrangement of Mg(2+) and pyrophosphate has not been reported in purine phosphoribosyltransferases. ImmucillinG in the binary complex is anchored by its 9-deazaguanine group, and the iminoribitol is disordered. No Mg(2+) or pyrophosphate is detected; thus, the 5'-phosphoryl group is needed to immobilize the iminoribitol prior to magnesium pyrophosphate binding. Filling the catalytic site involves (1) binding the purine ring, (2) anchoring the 5'-phosphate to fix the ribosyl group, (3) binding the first Mg(2+) to Asp125 and Glu126 carboxyl groups and binding Mg(2+).pyrophosphate, and (4) closing the catalytic site loop and formation of bound (Mg(2+))(2). pyrophosphate prior to catalysis. Guanine specificity is provided by two peptide carbonyl oxygens hydrogen-bonded to the exocyclic amino group and a weak interaction to O6. Transition state formation involves N7 protonation by Asp129 acting as the general acid.