Hydrophilically enhanced 3-carboranyl thymidine analogues (3CTAs) for boron neutron capture therapy (BNCT) of cancer

Five novel 3-carboranyl thymidine analogues (3CTAs) were designed and synthesized for boron neutron capture therapy (BNCT) of cancer. Phosphorylation of all five 3CTAs was catalyzed by recombinant human thymidine kinase (hTK1) using adenosine triphosphate (ATP) as the phosphate donor. The obtained phosphorylation rates ranged from 4% to 64.5% relative to that of thymidine. The compound with the most favorable hTK1 binding properties had a k(cat)/K(M) value of 57.4% relative to that of thymidine and an IC(50) of inhibition of thymidine phosphorylation by hTK1 of 92 microM. Among the five synthesized 3CTAs, this agent had also the overall most favorable physicochemical properties. Therefore, it may have the potential to replace N5-2OH, the current lead 3CTA, in preclinical studies. An in silico model for the binding of this compound to hTK1 was developed.     

Effect of Calcium Ions on Enteropeptidase Catalysis

The effects of calcium ions on hydrolysis of low molecular weight substrates catalyzed by different forms of enteropeptidase were studied. A method for determining activity of truncated enteropeptidase preparations lacking a secondary trypsinogen binding site and displaying low activity towards trypsinogen was developed using N-alpha-benzyloxycarbonyl-L-lysine thiobenzyl ester (Z-Lys-S-Bzl). The kinetic constants for hydrolysis of this substrate at pH 8.0 and 25 degrees C were determined for natural enteropeptidase (K(m) 59.6 microM, k(cat) 6660 min(-1), k(cat)/K(m) 111 microM(-1) x min(-1)), as well as for enteropeptidase preparation with deleted 118-783 fragment of the heavy chain (K(m) 176.9 microM, k(cat) 6694 min(-1), k(cat)/K(m) 37.84 microM(-1) x min(-1)) and trypsin (K(m) 56.0 microM, k(cat) 8280 min(-1), k(cat)/K(m) 147.86 microM(-1) x min(-1)). It was shown that the enzymes with trypsin-like primary active site display similar hydrolysis efficiency towards Z-Lys-S-Bzl. Calcium ions cause 3-fold activation of hydrolysis of the substrates of general type GD(4)K-X by the natural full-length enteropeptidase. In contrast, the hydrolysis of substrates with one or two Asp/Glu residues at P2-P3 positions is slightly inhibited by Ca2+. In the case of enteropeptidase light chain as well as the enzyme containing the truncated heavy chain (466-800 fragment), the activating effect of calcium ions was not detected for all the studied substrates. The results of hydrolysis experiments with synthetic enteropeptidase substrates GD(4)K-F(NO(2))G, G(5)DK-F(NO(2))G (where F(NO(2)) is p-nitrophenyl-L-phenylalanine residue), and GD(4)K-Nfa (where Nfa is beta-naphthylamide) demonstrate the possibility of regulation of undesired side hydrolysis using natural full-length enteropeptidase for processing chimeric proteins by means of calcium ions.     

Mechanistic analysis of wheat chlorophyllase

Chlorophyllase catalyzes the initial step in the degradation of chlorophyll and plays a key role in leaf senescence and fruit ripening. Here, we report the cloning of chlorophyllase from Triticum aestivum (wheat) and provide a detailed mechanistic analysis of the enzyme. Purification of recombinant chlorophyllase from an Escherichia coli expression system indicates that the enzyme functions as a dimeric protein. Wheat chlorophyllase hydrolyzed the phytol moiety from chlorophyll (k(cat) = 566 min(-1); K(m) = 63 microM) and was active over a broad temperature range (10-75 degrees C). In addition, the enzyme displays carboxylesterase activity toward p-nitrophenyl (PNP)-butyrate, PNP-decanoate, and PNP-palmitate. The pH-dependence of the reaction showed the involvement of an active site residue with a pK(a) of approximately 6.5 for both k(cat) and k(cat)/K(m) with chlorophyll, PNP-butyrate, and PNP-decanoate. Using these substrates, solvent kinetic isotope effects ranging from 1.5 to 1.9 and from 1.4 to 1.9 on k(cat) and k(cat)/K(m), respectively, were observed. Proton inventory experiments suggest the transfer of a single proton in the rate-limiting step. Our analysis of wheat chlorophyllase indicates that the enzyme uses a charge-relay mechanism similar to other carboxylesterases for catalysis. Understanding the activity and mechanism of chlorophyllase provides insight on the biological and chemical control of senescence in plants and lays the groundwork for biotechnological improvement of this enzyme.     

The inhibition of metallo-beta-lactamase by thioxo-cephalosporin derivatives

The 8-thioxocephalosporins are poor substrates for the B. cereus metallo beta-lactamase (k(cat)/K(m)=61.4M(-1) s(-1)) and act as weak competitive inhibitors (K(i) approximately 700 microM). The hydrolysis product of thioxocephalosporin, a thioacid, also inhibits the enzyme competitively with a K(i)=96 microM, whereas the cyclic thioxo-piperazinedione, formed by intramolecular aminolysis of thioxocephalexin has a K(i) of 29 microM.     

Re-engineering cytochrome P450 2B11dH for enhanced metabolism of several substrates including the anti-cancer prodrugs cyclophosphamide and ifosfamide

Based on recent directed evolution of P450 2B1, six P450 2B11 mutants at three positions were created in an N-terminal modified construct termed P450 2B11dH and characterized for enzyme catalysis using five substrates. Mutant I209A demonstrated a 3.2-fold enhanced k(cat)/K(m) for 7-ethoxy-4-trifluoromethylcourmarin O-deethylation, largely due to a dramatic decrease in K(m) (0.72 microM vs. 18 microM). I209A also demonstrated enhanced selectivity for testosterone 16beta-hydroxylation over 16alpha-hydroxylation. In contrast, V183L showed a 4-fold increased k(cat) for 7-benzyloxyresorufin debenzylation and a 4.7-fold increased k(cat)/K(m) for testosterone 16alpha-hydroxylation. V183L also displayed a 1.7-fold higher k(cat)/K(m) than P450 2B11dH with the anti-cancer prodrugs cyclophosphamide and ifosfamide, resulting from a approximately 4-fold decrease in K(m). Introduction of the V183L mutation into full-length P450 2B11 did not enhance the k(cat)/K(m). Overall, the re-engineered P450 2B11dH enzymes exhibited enhanced catalytic efficiency with several substrates including the anti-cancer prodrugs.     

Antioxidative function and substrate specificity of NAD(P)H-dependent alkenal/one oxidoreductase. A new role for leukotriene B4 12-hydroxydehydrogenase/15-oxoprostaglandin 13-reductase

There are several known routes for the metabolic detoxication of alpha,beta-unsaturated aldehydes and ketones, including conjugation to glutathione and reduction and oxidation of the aldehyde to an alcohol and a carboxylic acid, respectively. In this study, we describe a fourth class of detoxication that involves the reduction of the alpha,beta-carbon=carbon double bond to a single bond. This reaction is catalyzed by NAD(P)H-dependent alkenal/one oxidoreductase (AO), an enzyme heretofore known as leukotriene B4 12-hydroxydehydrogenase, 15-oxoprostaglandin 13-reductase, and dithiolethione-inducible gene-1. AO is shown to effectively reduce cytotoxic lipid peroxidation products such as 4-hydroxy-2-nonenal (HNE) (k(cat) = 4.0 x 10(3) min(-1); k(cat)/K(m) = 3.3 x 10(7) min(-1) M(-1)) and acrolein (k(cat) = 2.2 x 10(2) min(-1); k(cat)/K(m) = 1.5 x 10(6) min(-1) M(-1)) and common industrial compounds such as ethyl vinyl ketone (k(cat) = 9.6 x 10(3) min(-1); k(cat)/K(m) = 8.8 x 10(7) min(-1) M(-1)) and 15-oxoprostaglandin E1 (k(cat) = 2.4 x 10(3) min(-1); k(cat)/K(m) = 2.4 x 10(9) min(-1) M(-1)). Furthermore, transfection of human embryonic kidney cells with a rat liver AO expression vector protected these cells from challenge with HNE. The concentration of HNE at which 50% of the cells were killed after 24 h increased from approximately 15 microM in control cells to approximately 70 microM in AO-transfected cells. Overexpression of AO also completely abolished protein alkylation by HNE at all concentrations tested (up to 30 microM). Thus, we describe a novel antioxidative activity of a previously characterized bioactive lipid-metabolizing enzyme that could prove to be therapeutically or prophylactically useful due to its high catalytic rate and inducibility.     

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.     

Purification and characterization of Mn-peroxidase from Musa paradisiaca (banana) stem juice

Mn-peroxidase (MnP), a biotechnologically important enzyme was purified for the first time from a plant source Musa paradisiaca (banana) stem, which is an agro-waste easily available after harvest of banana fruits. MnP was earlier purified only from the fungal sources. The enzyme was purified from stem juice by ultrafiltration and anion-exchange column chromatography on diethylamino ethylcellulose with 8-fold purification and purification yield of 65%. The enzyme gave a single protein band in SDS-PAGE corresponding to molecular mass 43 kDa. The Native-PAGE of the enzyme also gave a single protein band, confirming the purity of the enzyme. The UV/VIS spectrum of the purified enzyme differed from the other heme peroxidases, as the Soret band was shifted towards lower wavelength and the enzyme had an intense absorption band around 250 nm. The K(m) values using MnSO4 and H2O2 as the substrates of the purified enzyme were 21.0 and 9.5 microM, respectively. The calculated k(cat) value of the purified enzyme using Mn(II) as the substrate in 50 mM lactate buffer (pH 4.5) at 25 degrees C was 6.7s(-1), giving a k(cat)/K(m) value of 0.32 microM(-1)s(-1). The k(cat) value for the MnP-catalyzed reaction was found to be dependent of the Mn(III) chelator molecules malonate, lactate and oxalate, indicating that the enzyme oxidized chelated Mn(II) to Mn(III). The pH and temperature optima of the enzyme were 4.5 and 25 degrees C, respectively. The enzyme in combination with H2O2 liberated bromine and iodine in presence of KBr and KI respectively. All these enzymatic characteristics were similar to those of fungal MnP. The enzyme has the potential as a green brominating and iodinating agent in combination with KBr/KI and H2O2.     

A detailed kinetic study of Mox-1, a plasmid-encoded class C beta-lactamase

Surveys of beta-lactamases in different parts of the world show an important increase in class C beta-lactamases, thus the study of these enzymes is becoming an important issue. We created an overproduction system for Mox-1, a plasmid class C beta-lactamase, by cloning the gene encoding this enzyme, and placing it under the control of a T7 promoter, using vector pET 28a. The enzyme, purified by ion exchange chromatography, was used to obtain the molecular mass (38246), the N-terminal sequence (GEASPVDPLRPVV), and pI (8.9), and to perform a detailed kinetic study. Cephalotin was used as reporter substrate in the case of poor substrates. The kinetic study showed that benzylpenicillin, cephalotin, cefcapene and moxalactam were good substrates for Mox-1 (k(cat)/K(m) values >2.5 x 10(6) M(-1) s(-1)). On the other hand, ceftazidime and cefepime were poor substrates for this enzyme (K(m) values >200 microM). Clavulanic acid had no inhibitory effect on Mox-1 (K(m)=30.2 mM), however aztreonam behaved as an inhibitor of Mox-1 (K(i)=2.85 microM).     

Neisseria gonorrhoeae penicillin-binding protein 3 exhibits exceptionally high carboxypeptidase and beta-lactam binding activities

A soluble form of penicillin-binding protein 3 (PBP 3) from Neisseria gonorrhoeae was expressed and purified from Escherichia coli and characterized for its interaction with beta-lactam antibiotics, its catalytic properties with peptide and peptidoglycan substrates, and its role in cell viability and morphology. PBP 3 had an unusually high k(2)/K' value relative to other PBPs for acylation with penicillin (7.7 x 10(5) M(-1) s(-1)) at pH 8.5 at 25 degrees C and hydrolyzed bound antibiotic very slowly (k(3) < 4.6 x 10(-5) s(-1), t(1/2) > 230 min). PBP 3 also demonstrated exceptionally high carboxypeptidase activity with a k(cat) of 580 s(-1) and a k(cat)/K(m) of 1.8 x 10(5) M(-1) s(-1) with the substrate N(alpha)-Boc-N(epsilon)-Cbz-L-Lys-D-Ala-D-Ala. This is the highest k(cat) value yet reported for a PBP or other serine peptidases. Activity against a approximately D-Ala-D-Lac peptide substrate was approximately 2-fold lower than against the analogous approximately D-Ala-D-Ala peptide substrate, indicating that deacylation is rate determining for both amide and ester hydrolysis. The pH dependence profiles of both carboxypeptidase activity and beta-lactam acylation were bell-shaped with maximal activity at pH 8.0-8.5. PBP 3 displayed weak transpeptidase activity in a model transpeptidase reaction but was active as an endopeptidase, cleaving dimeric peptide cross-links. Deletion of PBP 3 alone had little effect on viability, growth rate, and morphology of N. gonorrhoeae, although deletion of both PBP 3 and PBP 4, the other low-molecular-mass PBP in N. gonorrhoeae, resulted in a decreased growth rate and marked morphological abnormalities.     

Synthesis and biophysical evaluation of minor-groove binding C-terminus modified pyrrole and imidazole triamide analogs of distamycin

Five polyamide derivatives with rationally modified C-terminus moieties were synthesized and their DNA binding specificity and affinity determined. A convergent approach was employed to synthesize polyamides containing an alkylaminopiperazine (4 and 5), a truncated piperazine (6), or an alkyldiamino-C-terminus moiety (7 and 8) with two specific objectives: to investigate the effects of number of potential cationic centers and steric bulk at the C-terminus. CD studies confirmed that compounds 4, 5, 7, and 8 bind in the minor groove of DNA. The alkylpiperazine containing compounds (4 and 5) showed only moderate binding to DNA with DeltaT(m) values of 2.8 and 8.3 degrees C with their cognate sequence, respectively. The alkyldiamino compounds (7 and 8) were more impressive producing a DeltaT(m) of >17 and >22 degrees C, respectively. Compound 6 (truncated piperazine) did not stabilize its cognate DNA sequence. Footprints were observed for all compounds (except compound 6) with their cognate DNA sequence using DNase I footprinting, with compound 7 producing a footprint of 0.1 microM at the expected 5'-ACGCGT-3' site. SPR analysis of compound 7 binding to 5'-ACGCGT-3', 5'-ACCGGT-3', and 5'-AAATTT-3' produced binding affinities of 2.2x10(6), 3.3x10(5), and 1x10(5)M(-1), respectively, indicating a preference for its cognate sequence of 5'-ACGCGT-3'. These results are in good agreement with the footprinting data. The results indicate that steric crowding at the C-terminus is important with respect to binding. However, the number of cationic centers within the molecule may also play a role. The alkyldiamino-containing compounds (7 and 8) warrant further investigation in the field of polyamide research.     

Biochemical characterization of recombinant L-asparaginase (AnsA) from Rhizobium etli, a member of an increasing rhizobial-type family of L-asparaginases

We report the expression, purification, and characterization of L-asparaginase (AnsA) from Rhizobium etli. The enzyme was purified to homogeneity in a single-step procedure involving affinity chromatography, and the kinetic parameters K(m), V(max), and k(cat) for L-asparagine were determined. The enzymatic activity in the presence of a number of substrates and metal ions was investigated. The molecular mass of the enzyme was 47 kDa by SDS-PAGE. The enzyme showed a maximal activity at 50 degrees C, but the optimal temperature of activity was 37 degrees C. It also showed maximal and optimal activities at pH 9.0. The values of K(m), V(max), k(cat), and k(cat)/K(m) were 8.9 +/- 0.967 × 10?3 M, 128 +/- 2.8 U/mg protein, 106 +/- 2 s?1, and 1.2 +/- 0.105 × 10? M?1s?1, respectively. The L-asparaginase activity was reduced in the presence of Mn2?, Zn2?, Ca2?, and Mg2? metal ions for about 52% to 31%. In addition, we found that NH??, L-Asp, D-Asn, and beta-aspartyl-hydroxamate in the reaction buffer reduced the activity of the enzyme, whereas L-Gln did not modify its enzymatic activity. This is the first report on the expression and characterization of the L-asparaginase (AnsA) from R. etli. Phylogenetic analysis of asparaginases reveals an increasing group of known sequences of the Rhizobialtype asparaginase II family.     

Interactions of different inhibitors with active-site aspartyl residues of HIV-1 protease and possible relevance to pepsin

The importance of the active site region aspartyl residues 25 and 29 of the mature HIV-1 protease (PR) for the binding of five clinical and three experimental protease inhibitors [symmetric cyclic urea inhibitor DMP323, nonhydrolyzable substrate analog (RPB) and the generic aspartic protease inhibitor acetyl-pepstatin (Ac-PEP)] was assessed by differential scanning calorimetry. DeltaT(m) values, defined as the difference in T(m) for a given protein in the presence and absence of inhibitor, for PR with DRV, ATV, SQV, RTV, APV, DMP323, RPB, and Ac-PEP are 22.4, 20.8, 19.3, 15.6, 14.3, 14.7, 8.7, and 6.5 degrees C, respectively. Binding of APV and Ac-PEP is most sensitive to the D25N mutation, as shown by DeltaT(m) ratios [DeltaT(m)(PR)/DeltaT(m)(PR(D25N))] of 35.8 and 16.3, respectively, whereas binding of DMP323 and RPB (DeltaT(m) ratios of 1-2) is least affected. Binding of the substrate-like inhibitors RPB and Ac-PEP is nearly abolished (DeltaT(m)(PR)/DeltaT(m)(PR(D29N)) > or = 44) by the D29N mutation, whereas this mutation only moderately affects binding of the smaller inhibitors (DeltaT(m) ratios of 1.4-2.2). Of the nine FDA-approved clinical HIV-1 protease inhibitors screened, APV, RTV, and DRV competitively inhibit porcine pepsin with K(i) values of 0.3, 0.6, and 2.14 microM, respectively. DSC results were consistent with this relatively weak binding of APV (DeltaT(m) 2.7 degrees C) compared with the tight binding of Ac-PEP (DeltaT(m) > or = 17 degrees C). Comparison of superimposed structures of the PR/APV complex with those of PR/Ac-PEP and pepsin/pepstatin A complexes suggests a role for Asp215, Asp32, and Ser219 in pepsin, equivalent to Asp25, Asp25', and Asp29 in PR in the binding and stabilization of the pepsin/APV complex.     

Reactivity of toluate dioxygenase with substituted benzoates and dioxygen

Toluate dioxygenase (TADO) of Pseudomonas putida mt-2 catalyzes the dihydroxylation of a broad range of substituted benzoates. The two components of this enzyme were hyperexpressed and anaerobically purified. Reconstituted TADO had a specific activity of 3.8 U/mg with m-toluate, and each component had a full complement of their respective Fe(2)S(2) centers. Steady-state kinetics data obtained by using an oxygraph assay and by varying the toluate and dioxygen concentrations were analyzed by a compulsory order ternary complex mechanism. TADO had greatest specificity for m-toluate, displaying apparent parameters of KmA = 9 +/- 1 microM, k(cat) = 3.9 +/- 0.2 s(-1), and K(m)O(2) = 16 +/- 2 microM (100 mM sodium phosphate, pH 7.0; 25 degrees C), where K(m)O(2) represents the K(m) for O(2) and KmA represents the K(m) for the aromatic substrate. The enzyme utilized benzoates in the following order of specificity: m-toluate > benzoate approximately 3-chlorobenzoate > p-toluate approximately 4-chlorobenzoate > o-toluate approximately 2-chlorobenzoate. The transformation of each of the first five compounds was well coupled to O(2) utilization and yielded the corresponding 1,2-cis-dihydrodiol. In contrast, the transformation of ortho-substituted benzoates was poorly coupled to O(2) utilization, with >10 times more O(2) being consumed than benzoate. However, the apparent K(m) of TADO for these benzoates was >100 microM, indicating that they do not effectively inhibit the turnover of good substrates.     

Diffusion-limited component of reactions catalyzed by Bacillus cereus beta-lactamase I

The Bacillus cereus beta-lactamase I catalyzes the hydrolysis of a wide variety of penicillins and cephalosporins with values of k(cat)/K(m) varying over several orders of magnitude. The values of this parameter for the most reactive of these compounds, benzylpenicillin, I, and furylacryloyl-penicillin, II (k(cat)/K(m) = 2.43 x 10(7) M(-1) s(-1) and 2.35 x 10(7) M(-1) s(-1), respectively, at pH 7.0 in potassium phosphate buffer containing 0.17 M KCl, I(c) = 0.63, 25 degrees C) are decreased markedly by increasing viscosity in sucrose- or glycerol-containing buffers. The relative sensitivities to viscosity of k(cat)/K(m) values for I and for cephaloridine, III, were found to be virtually unchanged at pH 3.8 from those observed at pH 7.0. The differential effects of viscosity on the reactive vs. the sluggish [e.g., cephalothin (IV), k(cat)/K(m) = 1 x 10(4) M(-1) s(-1)] substrates support the contention that the rates of reaction of the former with the enzyme are in part diffusion controlled. Quantitative analysis gives values for the association rate constants, k(1), of 7.6 x 10(7) M(-1) s(-1), 4 x 10(7) M(-1) s(-1), and 1.1 x 10(7) M(-1) s(-1) for I, II, and III, respectively. As both reactive and sluggish substrates associate with the active site of the enzyme with relatively similar rate constants, the variation in k(cat)/K(m) values is primarily due to the variation in the partition ratios k(-1)/k(2), for the ES complex, which are 2.3, 0.77, and 30 for I, II, and III, respectively. The preceding analysis is based on direct application of the Stokes-Einstein diffusion law to enzyme kinetics. The range of applicability of this law to the diffusion of substrate size molecules and the mechanics of diffusion of ionic species through viscous solutions of sucrose vs. polymers are explored.     

Tight binding of deoxyribonucleotide triphosphates to human thymidine kinase 2 expressed in Escherichia coli. Purification and partial characterization of its dimeric and tetrameric forms

Human thymidine kinase 2 (hTK2) phosphorylates pyrimidine deoxyribonucleosides to the corresponding nucleoside monophosphates, using a nucleotide triphosphate as a phosphate donor. In this study, hTK2 was cloned and expressed at high levels in Escherichia coli as a fusion protein with maltose-binding protein. Induction of a heat-shock response by ethanol and coexpression of plasmid-encoded GroEL/ES chaperonins at 28 degrees C minimized the nonspecific aggregation of the hybrid protein and improved the recovery of three homooligomeric forms of the properly folded enzyme, i.e., dimer > tetramer > hexamer. The dimer and the tetramer were isolated in stable and highly purified forms after proteolytic removal of the fusion partner. Both oligomers contained a substoichiometric amount of deoxyribonucleotide triphosphates (dTTP > dCTP > dATP), known to be strong feedback inhibitors of the enzyme. Steady-state kinetic studies were consistent with the presence of endogenous inhibitors, and both oligomeric forms revealed a lag phase of at least approximately 5 min, which was abolished on preincubation with substrate (dThd or dCyd). The rather similar kinetic properties of the two oligomeric forms indicate that the basic functional unit is a dimer. Molecular docking experiments with a modeled hTK2 three-dimensional structure accurately predicted the binding positions at the active site of the natural substrates (dThd, dCyd, and ATP) and inhibitors (dTTP and dCTP), with highly conserved orientations obtained for all ligands. The calculated relative nonbonded interaction energies are in agreement with the biochemical data and show that the inhibitor complexes have lower stabilization energies (higher affinity) than the substrates.     

Natural substrate assay for chitinases using high-performance liquid chromatography: a comparison with existing assays

The determination of kinetic parameters of chitinases using natural substrates is difficult due to low K(m) values, which require the use of low substrate concentrations that are hard to measure. Using the natural substrate (GlcNAc)(4), we have developed an assay for the determination of k(cat) and K(m)values of chitinases. Product concentrations as low as 0.5 microM were detected using normal-phase high-performance liquid chromatography (HPLC) with an amide 80 column (0.20 x 25 cm) using spectrophotometric detection at 210 nm. By means of this assay, k(cat) and K(m)values for chitinases A (ChiA) and B (ChiB) of Serratia marcescens were found to be 33+/-1s(-1) and 9+/-1 microM and 28+/-2s(-1) and 4+/-2 microM, respectively. For ChiB, these values were compared to those found with commonly used substrates where the leaving group is a (nonnatural) chromophore, revealing considerable differences. For example, assays with 4-methylumbelliferyl-(GlcNAc)(2) yielded a k(cat) value of 18+/-2s(-1) and a K(m) value of 30+/-6 microM. For two ChiB mutants containing a Trp --> Ala mutation in the +1 or +2 subsites, the natural substrate and the 4-methylumbelliferyl-(GlcNAc)(2) assays yielded rather similar K(m) values (5-fold difference at most) but showed dramatic differences in k(cat) values (up to 90-fold). These results illustrate the risk of using artificial substrates for characterization of chitinases and, thus, show that the new HPLC-based assay is a valuable tool for future chitinase research.     

Cloning and characterization of histamine dehydrogenase from Nocardioides simplex

Histamine dehydrogenase (NSHADH) can be isolated from cultures of Nocardioides simplex grown with histamine as the sole nitrogen source. A previous report suggested that NSHADH might contain the quinone cofactor tryptophan tryptophyl quinone (TTQ). Here, the hdh gene encoding NSHADH is cloned from the genomic DNA of N. simplex, and the isolated enzyme is subjected to a full spectroscopic characterization. Protein sequence alignment shows NSHADH to be related to trimethylamine dehydrogenase (TMADH: EC 1.5.99.7), where the latter contains a bacterial ferredoxin-type [4Fe-4S] cluster and 6-S-cysteinyl FMN cofactor. NSHADH has no sequence similarity to any TTQ containing amine dehydrogenases. NSHADH contains 3.6+/-0.3 mol Fe and 3.7+/-0.2 mol acid labile S per subunit. A comparison of the UV/vis spectra of NSHADH and TMADH shows significant similarity. The EPR spectrum of histamine reduced NSHADH also supports the presence of the flavin and [4Fe-4S] cofactors. Importantly, we show that NSHADH has a narrow substrate specificity, oxidizing only histamine (K(m)=31+/-11 microM, k(cat)/K(m)=2.1 (+/-0.4)x10(5)M(-1)s(-1)), agmatine (K(m)=37+/-6 microM, k(cat)/K(m)=6.0 (+/-0.6)x10(4)M(-1)s(-1)), and putrescine (K(m)=1280+/-240 microM, k(cat)/K(m)=1500+/-200 M(-1)s(-1)). A kinetic characterization of the oxidative deamination of histamine by NSHADH is presented that includes the pH dependence of k(cat)/K(m) (histamine) and the measurement of a substrate deuterium isotope effect, (D)(k(cat)/K(m) (histamine))=7.0+/-1.8 at pH 8.5. k(cat) is also pH dependent and has a reduced substrate deuterium isotope of (D)(k(cat))=1.3+/-0.2.     

Steady-state kinetic characterization of substrates and metal-ion specificities of the full-length and N-terminally truncated recombinant human methionine aminopeptidases (type 2)

The steady-state kinetics of a full-length and truncated form of the type 2 human methionine aminopeptidase (hMetAP2) were analyzed by continuous monitoring of the amide bond cleavage of various peptide substrates and methionyl analogues of 7-amido-4-methylcoumarin (AMC) and p-nitroaniline (pNA), utilizing new fluorescence-based and absorbance-based assay substrates and a novel coupled-enzyme assay method. The most efficient substrates for hMetAP2 appeared to be peptides of three or more amino acids for which the values of k(cat)/K(m) were approximately 5 x 10(5) M(-1) min(-1). It was found that while the nature of the P1' residue of peptide substrates dictates the substrate specificity in the active site of hMetAP2, the P2' residue appears to play a key role in the kinetics of peptidolysis. The catalytic efficiency of dipeptide substrates was found to be at least 250-fold lower than those of the tripeptides. This substantially diminished catalytic efficiency of hMetAP2 observed with the alternative substrates MetAMC and MetpNA is almost entirely due to the reduction in the turnover rate (k(cat)), suggesting that cleavage of the amide bond is at least partially rate-limiting. The 107 N-terminal residues of hMetAP2 were not required for either the peptidolytic activity of the enzyme or its stability. Steady-state kinetic comparison and thermodynamic analyses of an N-terminally truncated form and full-length enzyme yielded essentially identical kinetic behavior and physical properties. Addition of exogenous Co(II) cation was found to significantly activate the full-length hMetAP2, while Zn(II) cation, on the other hand, was unable to activate hMetAP2 under any concentration that was tested.     

Diversity in the oxidation of substrates by cytochrome P450 2D6: lack of an obligatory role of aspartate 301-substrate electrostatic bonding

Cytochrome P450 (P450) 2D6 was first identified as the polymorphic human debrisoquine hydroxylase and subsequently shown to catalyze the oxidation of a variety of drugs containing a basic nitrogen. Residue Asp301 has been characterized as being involved in electrostatic interactions with substrates on the basis of homology modeling and site-directed mutagenesis experiments [Ellis, S. W., Hayhurst, G. P., Smith, G., Lightfoot, T., Wong, M. M. S., Simula, A. P., Ackland, M. J., Sternberg, M. J. E., Lennard, M. S., Tucker, G. T., and Wolf, C. R. (1995) J. Biol. Chem. 270, 29055-29058]. However, pharmacophore models based on the role of Asp301 in substrate binding are compromised by reports of catalytic activity toward substrates devoid of a basic nitrogen, which have generally been ignored. We characterized a high-affinity ligand for P450 2D6, also devoid of a basic nitrogen atom, spirosulfonamide [4-[3-(4-fluorophenyl)-2-oxo-1-oxaspiro[4.4]non-3-en-4-yl]benzenesulfonamide], with K(s) 1.6 microM. Spirosulfonamide is a substrate for P450 2D6 (k(cat) 6.5 min(-)(1) for the formation of a syn spiromethylene carbinol, K(m) 7 microM). Mutation of Asp301 to neutral residues (Asn, Ser, Gly) did not substantially affect the binding of spirosulfonamide (K(s) 2.5-3.5 microM). However, the hydroxylation of spirosulfonamide was attenuated in these mutants to the same extent (90%) as for the classic nitrogenous substrate bufuralol, and the effect of the D301N substitution was manifested on k(cat) but not K(m). Analogues of spirosulfonamide were also evaluated as ligands and substrates. Analogues in which the sulfonamide moiety was modified to an amide, thioamide, methyl sulfone, or hydrogen were ligands with K(s) values of 1.7-32 microM. All were substrates, and the methyl sulfone analogue was oxidized to the syn spiromethylene carbinol analogue of the major spirosulfonamide product. The D301N mutation produced varying changes in the oxidation patterns of the spirosulfonamide analogues. The peptidometic ritonavir and the steroids progesterone and testosterone had been reported to be substrates for P450 2D6, but the affinities (K(s)) were unknown; these were estimated to be 1.2, 1.5, and 15 microM, respectively (cf. 6 microM for the classic substrate bufuralol). The results are consistent with a role of Asp301 other than electrostatic interaction with a positively charged ligand. H-Bonding or electrostatic interactions probably enhance binding of some substrates, but our results show that it is not required for all substrates and explain why predictive models fail to recognize the proclivity for many substrates, especially those containing no basic nitrogen.