Structural insight into mechanism and diverse substrate selection strategy of L-ribulokinase

The araBAD operon encodes three different enzymes required for catabolism of L-arabinose, which is one of the most abundant monosaccharides in nature. L-ribulokinase, encoded by the araB gene, catalyzes conversion of L-ribulose to L-ribulose-5-phosphate, the second step in the catabolic pathway. Unlike other kinases, ribulokinase exhibits diversity in substrate selectivity and catalyzes phosphorylation of all four 2-ketopentose sugars with comparable k(cat) values. To understand ribulokinase recognition and phosphorylation of a diverse set of substrates, we have determined the X-ray structure of ribulokinase from Bacillus halodurans bound to L-ribulose and investigated its substrate and ATP co-factor binding properties. The polypeptide chain is folded into two domains, one small and the other large, with a deep cleft in between. By analogy with related sugar kinases, we identified (447)GGLPQK(452) as the ATP-binding motif within the smaller domain. L-ribulose binds in the cleft between the two domains via hydrogen bonds with the side chains of highly conserved Trp126, Lys208, Asp274, and Glu329 and the main chain nitrogen of Ala96. The interaction of L-ribulokinase with L-ribulose reveals versatile structural features that help explain recognition of various 2-ketopentose substrates and competitive inhibition by L-erythrulose. Comparison of our structure to that of the structures of other sugar kinases revealed conformational variations that suggest domain-domain closure movements are responsible for establishing the observed active site environment.     

Selective neurotensin-derived internally quenched fluorogenic substrates for neurolysin (EC 3.4.24.16): comparison with thimet oligopeptidase (EC 3.4.24.15) and neprilysin (EC 3.4.24.11)

Internally quenched fluorescent peptides derived from neurotensin (pELYENKPRRPYIL) sequence were synthesized and assayed as substrates for neurolysin (EC 3.4.24.16), thimet oligopeptidase (EC 3.4.24.15 or TOP), and neprilysin (EC 3.4.24.11 or NEP). Abz-LYENKPRRPYILQ-EDDnp (where EDDnp is N-(2,4-dinitrophenyl)ethylenediamine and Abz is ortho-aminobenzoic acid) was derived from neurotensin by the introduction of Q-EDDnp at the C-terminal end of peptide and by the substitution of the pyroglutamic (pE) residue at N-terminus for Abz and a series of shorter peptides was obtained by deletion of amino acids residues from C-terminal, N-terminal, or both sides. Neurolysin and TOP hydrolyzed the substrates at P--Y or Y--I or R--R bonds depending on the sequence and size of the peptides, while NEP cleaved P-Y or Y-I bonds according to its S'(1) specificity. One of these substrates, Abz-NKPRRPQ-EDDnp was a specific and sensitive substrate for neurolysin (k(cat) = 7.0 s(-1), K(m) = 1.19 microM and k(cat)/K(m) = 5882 mM(-1). s(-1)), while it was completely resistant to NEP and poorly hydrolyzed by TOP and also by prolyl oligopeptidase (EC 3.4.21.26). Neurolysin concentrations as low as 1 pM were detected using this substrate under our conditions and its analogue Abz-NKPRAPQ-EDDnp was hydrolyzed by neurolysin with k(cat) = 14.03 s(-1), K(m) = 0.82 microM, and k(cat)/K(m) = 17,110 mM(-1). s(-1), being the best substrate so far described for this peptidase.     

Characterization of a mannose-6-phosphate isomerase from Thermus thermophilus and increased L-ribose production by its R142N mutant

An uncharacterized gene from Thermus thermophilus, thought to encode a mannose-6-phosphate isomerase, was cloned and expressed in Escherichia coli. The maximal activity of the recombinant enzyme for L-ribulose isomerization was observed at pH 7.0 and 75°C in the presence of 0.5 mM Cu(2+). Among all of the pentoses and hexoses evaluated, the enzyme exhibited the highest activity for the conversion of L-ribulose to L-ribose, a potential starting material for many L-nucleoside-based pharmaceutical compounds. The active-site residues, predicted according to a homology-based model, were separately replaced with Ala. The residue at position 142 was correlated with an increase in L-ribulose isomerization activity. The R142N mutant showed the highest activity among mutants modified with Ala, Glu, Tyr, Lys, Asn, or Gln. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose using the R142N mutant were 1.4- and 1.6-fold higher than those of the wild-type enzyme, respectively. The k(cat)/K(m) of the R142N mutant was 3.8-fold higher than that of Geobacillus thermodenitrificans mannose-6-phosphate isomerase, which exhibited the highest activity to date for the previously reported k(cat)/K(m). The R142N mutant enzyme produced 213 g/liter L-ribose from 300 g/liter L-ribulose for 2 h, with a volumetric productivity of 107 g liter(-1) h(-1), which was 1.5-fold higher than that of the wild-type enzyme.     

Enzyme-coupled assay for beta-xylosidase hydrolysis of natural substrates

We describe here a new enzyme-coupled assay for the quantitation of d-xylose using readily available enzymes that allows kinetic evaluation of hemicellulolytic enzymes using natural xylooligosaccharide substrates. Hydrogen peroxide is generated as an intermediary analyte, which allows flexibility in the choice of the chromophore or fluorophore used as the final reporter. Thus, we present d-xylose quantitation results for solution-phase assays performed with both the fluorescent reporter resorufin, generated from N-acetyl-3,7-dihydroxyphenoxazine (Amplex Red), and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS), whose corresponding radical cation has an absorbance maximum at approximately 400 nm. We also describe a useful solid-phase variation of the assay performed with the peroxidase substrate 3,3'-diaminobenzidine tetrahydrochloride, which produces an insoluble brown precipitate. In addition, kinetic parameters for hydrolysis of the natural substrates xylobiose and xylotriose were obtained using this assay for a glycosyl hydrolase family 39 beta-xylosidase from Thermoanaerobacterium sp. strain JW/SL YS485 (Swiss-Prot accession no. O30360). At higher xylobiose substrate concentrations the enzyme showed an increase in the rate indicative of transglycosylation, while for xylotriose marked substrate inhibition was observed. At lower xylobiose concentrations k(cat) was 2.7 +/- 0.4 s(-1), K(m) was 3.3 +/- 0.7 mM, and k(cat)/K(m) was 0.82 +/- 0.21 mM(-1) . s(-1). Nonlinear curve fitting to a substrate inhibition model showed that for xylotriose K(i) was 1.7 +/- 0.1 mM, k(cat) was 2.0 +/- 0.1 s(-1), K(m) was 0.144 +/- 0.011 mM, and k(cat)/K(m) was 14 +/- 1.3 mM(-1) . s(-1).     

Production of L-ribose from L-ribulose by a triple-site variant of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans

A triple-site variant (W17Q N90A L129F) of mannose-6-phosphate isomerase from Geobacillus thermodenitrificans was obtained by combining variants with residue substitutions at different positions after random and site-directed mutagenesis. The specific activity and catalytic efficiency (k(cat)/K(m)) for L-ribulose isomerization of this variant were 3.1- and 7.1-fold higher, respectively, than those of the wild-type enzyme at pH 7.0 and 70°C in the presence of 1 mM Co(2+). The triple-site variant produced 213 g/liter l-ribose from 300 g/liter L-ribulose for 60 min, with a volumetric productivity of 213 g liter(-1) h(-1), which was 4.5-fold higher than that of the wild-type enzyme. The k(cat)/K(m) and productivity of the triple-site variant were approximately 2-fold higher than those of the Thermus thermophilus R142N variant of mannose-6-phosphate isomerase, which exhibited the highest values previously reported.     

Rhodobacter capsulatus 1-deoxy-D-xylulose 5-phosphate synthase: steady-state kinetics and substrate binding

1-Deoxy-d-xylulose 5-phosphate synthase (DXP synthase) catalyzes the thiamine diphosphate (TPP)-dependent condensation of pyruvate and d-glyceraldehyde 3-phosphate (GAP) to yield DXP in the first step of the methylerythritol phosphate pathway for isoprenoid biosynthesis. Steady-state kinetic constants for DXP synthase calculated from the initial velocities measured at varying concentrations of substrates were as follows: k(cat) = 1.9 +/- 0.1 s(-1), K(m)(GAP) = 0.068 +/- 0.001 mM, and K(m)(pyruvate) = 0.44 +/- 0.05 mM for pyruvate and GAP; k(cat) = 1.7 +/- 0.1 s(-1), K(m)(d-glyceraldehyde) = 33 +/- 3 mM, and K(m)(pyruvate) = 1.9 +/- 0.5 mM for d-glyceraldehyde and pyruvate. beta-Fluoropyruvate was investigated as a dead-end inhibitor for pyruvate. Double-reciprocal plots showed a competitive inhibition pattern with respect to pyruvate and noncompetitive inhibition with respect to GAP/d-glyceraldehyde. (14)CO(2) trapping experiments demonstrated that the binding of both substrates (pyruvate and GAP/d-glyceraldehyde) is required for the formation of a catalytically competent enzyme-substrate complex. These results are consistent with an ordered mechanism for DXP synthase where pyruvate binds before GAP/d-glyceraldehyde.     

Human bile salt-dependent lipase efficiency on medium-chain acyl-containing substrates: control by sodium taurocholate

Bile salt-dependent lipase was purified to homogeneity from lyophilized human milk and used to screen the influence of the acyl chain length (2-16 carbon atoms) on the kinetic constants k(cat) and K(m) of the hydrolysis of para-nitrophenyl (pnp) ester substrates in the presence or absence of sodium taurocholate (NaTC: 0.02-20 mM). The highest k(cat) value (～3,500 s(-1)) was obtained with pnpC(8) as substrate, whereas the lowest K(m) (<10 μM) was that recorded with pnpC(10). In the absence of NaTC, the maximal catalytic efficiency (k(cat)/K(m)) was obtained with pnpC(8), while in the presence of NaTC k(cat)/K(m) was maximal with pnpC(8), pnpC(10) or pnpC(12). The bile salt activated the enzyme in two successive saturation phases occurring at a micromolar and a millimolar concentration range, respectively. The present data emphasize the suitability of this enzyme for the hydrolysis of medium-chain acyl-containing substrates and throw additional light on how BSDL is activated by NaTC.     

pH dependence and solvent deuterium oxide kinetic isotope effects on Bacillus cereus beta-lactamase I catalyzed reactions

The solvent kinetic isotope effects (SKIE's) on k(cat) (D(V)) and on k(cat/Km[D(V/K)] were determined for the Bacillus cereus beta-lactamase I catalyzed hydrolysis of five substrates that have values of k(cat)/K(m) varying over the range (0.014-46.3) X 10(6)M(-1) s(-1) and of k(cat) between 0.5 and 2019 s(-1). The variation of D(V/K) was only from 1.06 to 1.25 among these compounds and that in D(V) was from 1.50 to 2.16. These results require that Dk(1), the SKIE on the enzyme-substrate association rate constant, and D(k-1/k2), that on the partition ratio of the ES complex, both be near 1. The larger SKIE observed on D(V) requires that an exchangeable proton be in flight for either or both the acylation and the deacylation reaction. The pH dependence of the values k(cat)/K(m) for three substrates shows identical pK(a)s of 5.5. and 8.4. This identity combined with the fact that only one of these three substrates is kinetically "sticky" proves that the substrates can combine productively with only one protonic form of the enzyme. There is considerable substrate variation in the pK(a) values of k(cat) observed vs. pH profiles; the inflection points for all substrates studied are at pH values more extreme than are observed in the pH profiles for k(cat)/K(m).     

The Streptomyces-produced antibiotic fosfomycin is a promiscuous substrate for archaeal isopentenyl phosphate kinase

Isopentenyl phosphate kinase (IPK) catalyzes the phosphorylation of isopentenyl phosphate to form the isoprenoid precursor isopentenyl diphosphate in the archaeal mevalonate pathway. This enzyme is highly homologous to fosfomycin kinase (FomA), an antibiotic resistance enzyme found in a few strains of Streptomyces and Pseudomonas whose mode of action is inactivation by phosphorylation. Superposition of Thermoplasma acidophilum (THA) IPK and FomA structures aligns their respective substrates and catalytic residues, including H50 and K14 in THA IPK and H58 and K18 in Streptomyces wedmorensis FomA. These residues are conserved only in the IPK and FomA members of the phosphate subdivision of the amino acid kinase family. We measured the fosfomycin kinase activity of THA IPK [K(m) = 15.1 ± 1.0 mM, and k(cat) = (4.0 ± 0.1) × 10?2 s?1], resulting in a catalytic efficiency (k(cat)/K(m) = 2.6 M?1 s?1) that is 5 orders of magnitude lower than that of the native reaction. Fosfomycin is a competitive inhibitor of IPK (K(i) = 3.6 ± 0.2 mM). Molecular dynamics simulation of the IPK·fosfomycin·MgATP complex identified two binding poses for fosfomycin in the IP binding site, one of which results in a complex analogous to the native IPK·IP·ATP complex that engages H50 and the lysine triangle formed by K5, K14, and K205. The other binding pose leads to a dead-end complex that engages K204 near the IP binding site to bind fosfomycin. Our findings suggest a mechanism for acquisition of FomA-based antibiotic resistance in fosfomycin-producing organisms.     

The use of deoxyfluoro-d-glucopyranoses and related compounds in a study of yeast hexokinase specificity

1. 2-Deoxy-2-fluoro-d-glucose, 2-deoxy-2-fluoro-d-mannose and 2-deoxy-2,2-difluoro-d-arabino-hexose are good substrates for yeast hexokinase. 2. 3-Deoxy-3-fluoro-d-glucose and 4-deoxy-4-fluoro-d-glucose are poor substrates and have very similar K(m) values (8x10(-2)m). 3. Neither alpha- nor beta-d-glucopyranosyl fluoride is a substrate or inhibitor. 4. Studies with 2-chloro-2-deoxy- and 2-O-methyl derivatives of d-glucose and d-mannose have revealed that little chemical modification is possible at position 2 without substantial loss in substrate binding. 5. The variation in the value of K(m) for the d-hexose derivatives was associated with a corresponding change in the value of K(m) for MgATP(2-) showing that the binding of MgATP(2-) is modified by the binding of the sugar.     

The interaction of the phosphonate analogue of 3-phospho-d-glycerate with phosphoglycerate kinase

The methylene analogue of 3-phospho-d-glycerate, 2-hydroxy-4-phosphono-dl-butyric acid, is a substrate for phosphoglycerate kinase. The pK(a) values for the final dissociation of the natural substrate and its methylene isostere are 6.20 and 7.45 respectively. The kinetic parameters K(m) and k(cat.) for the enzyme-catalysed reaction were determined at pH6.9 and 8.5 by using low substrate concentrations. Although the k(cat.) values for the two substrates at each pH are similar, there is a 60-fold increase in the K(m) value for the methylene isostere on going to the lower pH. The results are most readily interpreted in terms of a dianionic group on C-3 being required for efficient substrate binding to the enzyme.     

Conversion of methylamine dehydrogenase to a long-chain amine dehydrogenase by mutagenesis of a single residue

Methylamine dehydrogenase (MADH) is a tryptophan tryptophylquinone (TTQ) dependent enzyme that catalyzes the oxidative deamination of primary amines. Amino acid residues of both the TTQ-bearing beta subunit and the noncatalytic alpha subunit line a substrate channel that leads from the protein surface to the enzyme active site. Phe55 of the alpha subunit is located at the opening of the active site. Conversion of alphaPhe55 to alanine dramatically alters the substrate preference of MADH. The K(m) for methylamine increases from 9 microM to 15 mM. The preferred substrates are now primary amines with chain lengths of at least seven carbons. The K(m) for 1, 10-diaminodecane is 11 microM, compared to 1.2 mM for wild-type MADH. Despite the large variation in K(m) values, k(cat) values are relatively unaffected by the mutation. Molecular modeling of substrates into the crystal structure of the enzyme active site and substrate channel provides an explanation for the dramatic changes in substrate specificity caused by this mutation of a single amino acid residue.     

Synthesis and hydrolysis by cysteine and serine proteases of short internally quenched fluorogenic peptides

We developed sensitive substrates for cysteine proteases and specific substrates for serine proteases based on short internally quenched fluorescent peptides, Abz-F-R-X-EDDnp, where Abz (ortho-aminobenzoic acid) is the fluorescent donor, EDDnp [N-(ethylenediamine)-2,4-dinitrophenyl amide] is the fluorescent quencher, and X are natural amino acids. This series of peptides is compared to the commercially available Z-F-R-MCA, where Abz and X replace carbobenzoxy (Z) and methyl-7-aminocoumarin amide (MCA), respectively; and EDDnp can be considered a P(2)' residue. Whereas MCA is the fluorescent probe and cannot be modified, in the series Abz-F-R-X-EDDnp the amino acids X give the choice of matching the specificity of the S(1)' enzyme subsite, increasing the substrate specificity for a particular protease. All Abz-F-R-X-EDDnp synthesized peptides (for X = Phe, Leu, Ile, Ala, Pro, Gln, Ser, Lys, and Arg) were assayed with papain, human cathepsin L and B, trypsin, human plasma, and tissue kallikrein. Abz-F-R-L-EDDnp was the best substrate for papain and Abz-F-R-R-EDDnp or Abz-F-R-A-EDDnp was the more susceptible to cathepsin L. Abz-F-R-L-EDDnp was able to detect papain in the range of 1 to 15 pM. Human plasma kallikrein hydrolyzed Abz-F-R-R-EDDnp with significant efficiency (k(cat)/K(m) = 1833 mM(-1) s(-1)) and tissue kallikrein was very selective, hydrolyzing only the peptides Abz-F-R-A-EDDnp (k(cat)/K(m) = 2852 mM(-1) s(-1)) and Abz-F-R-S-EDDnp (k(cat)/K(m) = 4643 mM(-1) s(-1)). All Abz-F-R-X-EDDnp peptides were resistant to hydrolysis by thrombin and activated factor X.     

The use of deoxyfluoro-d-galactopyranoses in a study of yeast galactokinase specificity

1. 2-Deoxy-2-fluoro-d-galactose, 3-deoxy-3-fluoro-d-galactose, 4-deoxy-4-fluoro-d-galactose, 6-deoxy-6-fluoro-d-galactose and 2-deoxy-d-lyxo-hexose are substrates for yeast galactokinase. 2. The variation in K(m) values for the d-hexose derivatives was not associated with a variation in the value of K(m) for MgATP(2-) indicating that the binding of MgATP(2-) is not modified by the binding of the sugar substrate. 3. Donated H bonds from OH-3, OH-4 and OH-6 and an accepted H bond to OH-2 of the d-hexose are important for the binding of the sugar substrate to galactokinase. 4. Yeast galactokinase exhibits similar kinetics to the galactokinase from Escherichia coli and operates by a similar random sequential mechanism. 5. 4-Deoxy-4-fluoro-d-glucose was neither a substrate for nor an inhibitor of yeast galactokinase.     

A novel, arsenite-sensitive E2 of the ubiquitin pathway: purification and properties

In the multienzyme ubiquitin-dependent proteolytic pathway, conjugation of ubiquitin to target proteins serves as a signal for protein degradation. Rabbit reticulocytes possess a family of proteins, known as E2's, that form labile ubiquitin adducts by undergoing transthiolation with the ubiquitin thiol ester form of ubiquitin activating enzyme (E1). Only one E2 appears to function in ubiquitin-dependent protein degradation. The others have been postulated to function in regulatory ubiquitin conjugation. We have purified and characterized a previously undescribed E2 from rabbit reticulocytes. E2(230K) is an apparent monomer with a molecular mass of 230 kDa. The enzyme forms a labile ubiquitin adduct in the presence of E1, ubiquitin, and MgATP and catalyzes conjugation of ubiquitin to protein substrates. Exogenous protein substrates included yeast cytochrome c(Km = 125 mu M; kcat approximately 0.37 min-1) and histone H3 (Km less than 1.3 mu M; kcat approximately 0.18 min-1) as well as lysozyme, alpha-lactalbumin, and alpha-casein. E2(230K) did not efficiently reconstitute Ub-dependent degradation of substrates that it conjugated, either in the absence or in the presence of the ubiquitin-protein ligase that is involved in degradation. E2(230K) may thus be an enzyme that functions in regulatory Ub conjugation. Relative to other E2's, which are very iodoacetamide sensitive, E2(230K) was more slowly inactivated by iodoacetamide (k(obs) = 0.037 min-1 at 1.5 mM iodoacetamide; pH 7.0, 37 degrees C). E2(230K) was also unique among E2's in being subject to inactivation by inorganic arsenite (k(i)max = 0.12 min-1; K(0.5) = 3.3 mM; pH 7.0, 37 degrees C). Arsenite is considered to be a reagent specific for vicinal sulfhydryl sites in proteins, and inhibition is usually rapidly reversed upon addition of competitive dithiol compounds. Inactivation of E2(230K) by arsenite was not reversed within 10 min after addition of dithiothreitol at a concentration that blocked inactivation if it was premixed with arsenite; inactivation is therefore irreversible or very slowly reversible. We postulate that a conformation change of E2(230K) may be rate-limiting for interaction of enzyme thiol groups with arsenite.     

The role of the S1 binding site of carboxypeptidase M in substrate specificity and turn-over

The influence of the P1 amino acid on the substrate selectivity, the catalytic parameters K(m) and k(cat), of carboxypeptidase M (CPM) (E.C. 3.4.17.12) was systematically studied using a series of benzoyl-Xaa-Arg substrates. CPM had the highest catalytic efficiency (k(cat)/K(m)) for substrates with Met, Ala and aromatic amino acids in the penultimate position and the lowest with amino acids with branched side-chains. Substrates with Pro in P1 were not cleaved in similar conditions. The P1 substrate preference of CPM differed from that of two other members of the carboxypeptidase family, CPN (CPN/CPE subfamily) and CPB (CPA/CPB subfamily). Aromatic P1 residues discriminated most between CPM and CPN. The type of P2 residue also influenced the k(cat) and K(m) of CPM. Extending the substrate up to P7 had little effect on the catalytic parameters. The substrates were modelled in the active site of CPM. The results indicate that P1-S1 interactions play a role in substrate binding and turn-over.     

Screening for the optimal specificity profile of protein kinase C using electrospray mass-spectrometry

A peptide library approach based on electrospray mass-spectrometric (ESI-MS) detection of phosphopeptides was designed for rapid and quantitative characterization of protein kinase specificity. The k(cat)/K(m) values for the protein kinase Cbeta (PKCbeta) were determined for a systematically varied set of individual substrate peptides in library mixtures by the ESI-MS method. The analysis revealed a complex structural specificity profile in positions around the phosphorylated serine with hydrophobic and/or basic residues being mostly preferred. On the basis of the kinetic parameters, a highly efficient peptide substrate for PKCbeta (K(m)value below 100 nM) FRRRRSFRRR and its alanine substituted pseudosubstrate-analog inhibitor (K(i) value of 76 nM) were designed. The quantitative specificity profiles obtained by the new approach contained more information about kinase specificity than the conventional substrate consensus motifs. The new method presents a promising basis for design of substrate-site directed peptide or peptidomimetic inhibitors of protein kinases. Second, highly specific substrates could be designed for novel applications such as high-throughput protein kinase activity screens on protein kinase chips.     

Kinetics of substrate transglycosylation by glycoside hydrolase family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium

To elucidate the interaction between substrate inhibition and substrate transglycosylation of retaining glycoside hydrolases (GHs), a steady-state kinetic study was performed for the GH family 3 glucan (1-->3)-beta-glucosidase from the white-rot fungus Phanerochaete chrysosporium, using laminarioligosaccharides as substrates. When laminaribiose was incubated with the enzyme, a transglycosylation product was detected by thin-layer chromatography. The product was purified by size-exclusion chromatography, and was identified as a 6-O-glucosyl-laminaribiose (beta-D-Glcp-(1-->6)-beta-D-Glcp-(1-->3)-D-Glc) by 1H NMR spectroscopy and electrospray ionization mass spectrometry analysis. In steady-state kinetic studies, an apparent decrease of laminaribiose hydrolysis was observed at high concentrations of the substrate, and the plots of glucose production versus substrate concentration were thus fitted to a modified Michaelis-Menten equation including hydrolytic and transglycosylation parameters (K(m), K(m2), k(cat), k(cat2)). The rate of 6-O-glucosyl-laminaribiose production estimated by high-performance anion-exchange chromatography coincided with the theoretical rate calculated using these parameters, clearly indicating that substrate inhibition of this enzyme is fully explained by substrate transglycosylation. Moreover, when K(m), k(cat), and affinity for glucosyl-enzyme intermediates (K(m2)) were estimated for laminarioligosaccharides (DP=3-5), the K(m) value of laminaribiose was approximately 5-9 times higher than those of the other oligosaccharides (DP=3-5), whereas the K(m2) values were independent of the DP of the substrates. The kinetics of transglycosylation by the enzyme could be well interpreted in terms of the subsite affinities estimated from the hydrolytic parameters (K(m) and k(cat)), and a possible mechanism of transglycosylation is proposed.     

Dictyostelium discoideum salvages purine deoxyribonucleosides by highly specific bacterial-like deoxyribonucleoside kinases

The salvage of deoxyribonucleosides in the social amoeba Dictyostelium discoideum, which has an extremely A+T-rich genome, was investigated. All native deoxyribonucleosides were phosphorylated by D. discoideum cell extracts and we subcloned three deoxyribonucleoside kinase (dNK) encoding genes. D. discoideum thymidine kinase was similar to the human thymidine kinase 1 and was specific for thymidine with a K(m) of 5.1 microM. The other two cloned kinases were phylogenetically closer to bacterial deoxyribonucleoside kinases than to the eukaryotic enzymes. D. discoideum deoxyadenosine kinase (DddAK) had a K(m) for deoxyadenosine of 22.7 microM and a k(cat) of 3.7 s(-1) and could not efficiently phosphorylate any other native deoxyribonucleoside. D. discoideum deoxyguanosine kinase was also a purine-specific kinase and phosphorylated significantly only deoxyguanosine, with a K(m) of 1.4 microM and a k(cat) of 3 s(-1). The two purine-specific deoxyribonucleoside kinases could represent ancient enzymes present in the common ancestor of bacteria and eukaryotes but remaining only in a few eukaryote lineages. The narrow substrate specificity of the D. discoideum dNKs reflects the biased genome composition and we attempted to explain the strict preference of DddAK for deoxyadenosine by modeling the active center with different substrates. Apart from its native substrate, deoxyadenosine, DddAK efficiently phosphorylated fludarabine. Hence, DddAK could be used in the enzymatic production of fludarabine monophosphate, a drug used in the treatment of chronic lymphocytic leukemia.     

Substrate-based design of reversible Pin1 inhibitors

Human Pin1, a peptidyl-prolyl cis/trans isomerase with high specificity to -Ser/Thr(PO(3)H(2))-Pro- motifs, is required for cell cycle progression. In an effort to design reversible Pin1 inhibitors by using a substrate structure based approach, a panel of peptides were applied to systematically analyze the minimal structural requirements for Pin1 substrate recognition. Pin1 catalysis (k(cat)/K(m) < 5 mM(-1) s(-1)) for Ala-Pro, Ser-Pro, and Ser(PO(3)H(2))-Pro was detected using direct UV-visible spectrophotometric detection of prolyl isomerization, while weak competitive inhibition of Pin1 by these dipeptides was observed (K(i) > 1 mM). Substrates with chain lengths extending from either the P2 to P1' or the P1 to P2' subsite gave k(cat)/K(m) values of 100 mM(-1) s(-1) for Ala-Ser(PO(3)H(2))-Pro and 38 mM(-1) s(-1) for Ser(PO(3)H(2))-Pro-Arg. For both Pin1 and its yeast homologue Ess1, the optimal subsite recognition elements comprise five amino acid residues with the essential Ser(PO(3)H(2)) in the middle position. The resulting substrate Ac-Ala-Ala-Ser(PO(3)H(2))-Pro-Arg-NH-4-nitroanilide possesses a very low cis/trans interconversion barrier in the presence of either Pin1 or Ess1, with k(cat)/K(m) = 9300 mM(-1) s(-1) and 12000 mM(-1) s(-1), respectively. The D-Ser(PO(3)H(2)) residue preceding proline could serve as a substrate-deactivating determinant without compromising ground state affinity. Similarly, substitution of the amide bond preceding proline with a thioxo amide bond produces a potent inhibitor. Pin1 is reversibly inhibited by such substrate analogue inhibitors with IC(50) values in the low micromolar range. The D-amino acid containing inhibitor also exhibits remarkable stability against phosphatase activity in cell lysate.