Identification of the reactive histidine of cucumisin, a plant serine protease: modification with peptidyl chloromethyl ketone derivative of peptide substrate

A radioactive peptidyl chloromethyl ketone derived from substrates of cucumisin, 3H-labeled Z-Ala-Ala-Pro-Phe-chloromethyl ketone (3H-ZAAPFCK), was synthesized. When cucumisin was incubated with a 100-fold molar excess of 3H-ZAAPFCK for 16 h, 98% of the cucumisin activity was inhibited and about 0.93 mol of 3H-ZAAPFCK was incorporated in 1 mol of cucumisin. The 3H-ZAAPFCK-modified cucumisin was reduced and pyridylethylated, and then digested by trypsin. The radioactive peptide fragment was isolated and its amino acid sequence was determined. The radioactive fragment contained 32 amino acid residues and the sequence around the labeled residue was found to be -Asp-Thr-Asn-Gly-(His)-Gly-Thr-His-Thr-Ala-. This sequence is analogous to that around the reactive site histidine residue of the subtilisin family.     

Binding characteristics of SLE anti-DNA autoantibodies to modified DNA analogues

Native calf thymus DNA was modified with furocoumarins and reactive oxygen species (ROS). The modifications were probed by UV & Fluorescence spectroscopy, thermal denaturation and hydroxyapatite chromatography. In CD spectroscopy changes in the ellipticity of the DNA molecule were observed as a result of modification. The binding specificity of naturally occurring anti-DNA antibodies with modified DNA molecules was assessed by ELISA and quantitative precipitin titration. The affinity of anti-DNA antibodies for modified conformers was found to be quite high.     

Improved binding of cytochrome P450cam substrate analogues designed to fill extra space in the substrate binding pocket

Cytochrome P450cam catalyzes the 5-exo-hydroxylation of camphor. Camphor analogues were designed to fill an empty region of the substrate binding pocket with the expectation that they would bind more tightly than camphor itself due to increased van der Waals interactions with the protein and the displacement of any solvent occupying this site. A series of compounds (endo-borneol methyl ether, endo-borneol propyl ether, endo-borneol allyl ether and endo-borneol dimethyl allyl ether) were synthesized with substituents at the camphor carbonyl oxygen. The spin conversion and thermodynamic properties of this series of compounds were measured for wild type and Y96F mutant cytochrome P450cam and were interpreted in the context of molecular dynamics simulations of the camphor analogues in the P450 binding site and in solution. Compounds with a 3-carbon chain substituent were predicted to match the size of the unoccupied region most optimally and thus bind best. Consistent with this prediction, the borneol allyl ether binds to cytochrome P450cam with highest affinity with a Kd = 0.6 +/- 0.1 microM (compared to a Kd = 1.7 +/- 0.2 microM for camphor under the same experimental conditions). Binding of the camphor analogues to the Y96F mutant is much enhanced over the binding of camphor, indicating that hydrogen bonding plays a less important role in binding of these analogues. Binding enthalpies calculated from the simulations, taking all solvent contributions into account, agree very well with experimental binding enthalpies. Binding affinity is not however correlated with the calculated binding enthalpy because the binding of the substrate analogues is characterized by enthalpy-entropy compensation. The new compounds are useful probes for further studies of the mechanism of cytochrome P450cam due to their high binding affinities and high spin properties.     

Amino acids and peptides. XXXVI. Synthesis of enkephalin chloromethyl ketone and evaluation of its inhibitory activity against endopeptidase 22.19

Boc-Tyr-Gly-Gly-Phe-Leu-Ch2Cl was synthesized by the conventional solution method. During the course of acid hydrolysis (6N HCl, 110 degrees C, 18h) of Boc-Phe-Leu-CH2Cl, side reaction occurred, resulting in low recovery of Phe residue on amino acid analysis. The inhibitory activity of the synthesized Boc-Tyr-Gly-Gly-Phe-Leu-CH2Cl against endopeptidase 22.19, an enzyme related to the metabolism of opioid peptides, was examined.     

Calf spleen purine nucleoside phosphorylase complexed with substrates and substrate analogues

Purine nucleoside phosphorylase (PNP) is a key enzyme in the purine salvage pathway, which provides an alternative to the de novo pathway for the biosynthesis of purine nucleotides. PNP catalyzes the reversible phosphorolysis of 2'-deoxypurine ribonucleosides to the free bases and 2-deoxyribose 1-phosphate. Absence of PNP activity in humans is associated with specific T-cell immune suppression. Its key role in these two processes has made PNP an important drug design target. We have investigated the structural details of the PNP-catalyzed reaction by determining the structures of bovine PNP complexes with various substrates and substrate analogues. The preparation of phosphate-free crystals of PNP has allowed us to analyze several novel complexes, including the ternary complex of PNP, purine base, and ribose 1-phosphate and of the completely unbound PNP. These results provide an atomic view for the catalytic mechanism for PNP proposed by M. D. Erion et al. [(1997) Biochemistry 36, 11735-11748], in which an oxocarbenium intermediate is stabilized by phosphate and the negative charge on the purine base is stabilized by active site residues. The bovine PNP structure reveals several new details of substrate and inhibitor binding, including two phosphate-induced conformational changes involving residues 33-36 and 56-69 and a previously undetected role for His64 in phosphate binding. In addition, a well-ordered water molecule is found in the PNP active site when purine base or nucleoside is also present. In contrast to human PNP, only one phosphate binding site was observed. Although binary complexes were observed for nucleoside, purine base, or phosphate, ribose 1-phosphate binding occurs only in the presence of purine base.     

Binding, encapsulation and ejection: substrate dynamics during a chaperonin-assisted folding reaction

Mitochondrial malate dehydrogenase (mMDH) folds more rapidly in the presence of GroEL, GroES and ATP than it does unassisted. The increase in folding rate as a function of the concentration of GroEL-ES reaches a maximum at a stoichiometry which is approximately equimolar (mMDH subunits:GroEL oligomer) and with an apparent dissociation constant K' for the GroE acceptor state of at least 1 x 10(-8) M. However, even at chaperonin concentrations which are 4000 x K', i.e. at negligible concentrations of free mMDH, the observed folding rate of the substrate remains at its optimum, showing not only that folding occurs in the chaperonin-mMDH complex but also that this rate is uninhibited by any interactions with sites on GroEL. Despite the ability of mMDH to fold on the chaperonin, trapping experiments show that its dwell time on the complex is only 20 seconds. This correlates with both the rate of ATP turnover and the dwell time of GroES on the complex and is only approximately 5% of the time taken for the substrate to commit to the folded state. The results imply that ATP drives the chaperonin complex through a cycle of three functional states: (1) an acceptor complex in which the unfolded substrate is bound tightly; (2) an encapsulation state in which it is sequestered but direct protein-protein contact is lost so that folding can proceed unhindered; and (3) an ejector state which forces dissociation of the substrate whether folded or not.     

The behavior of substrate analogues and secondary deuterium isotope effects in the phenylalanine ammonia-lyase reaction

The present article reviews the results of experimental studies on paraquat neurotoxicity, started by our group several years ago--when clinical and experimental reports had increased the interest for the possibility that environmental chemicals, including paraquat, may be related to the development of Parkinson's disease-, and which are still continuing since paraquat appears to be a promising tool to study the mechanisms of neuronal cell death in vivo. Our observations have demonstrated that paraquat causes evident neurotoxic effects after intracerebroventricular or intracerebral injection in experimental animals; however, it seems that the herbicide does not exibit a selective neurotoxicity towards the dopaminergic nigro-striatal system since potent behavioural and electrocortical changes are induced by paraquat after injection in brain areas other than the substantia nigra and caudate nucleus. By studying the mechanisms through which paraquat induces neurotoxic effects in vivo, it was shown that either free radical production and activation of cholinergic and glutamatergic transmission may be regarded as related events which play a crucial role in paraquat-induced neurotoxicity. In addition, it was observed that in rats paraquat penetrates the blood-brain barrier following systemic administration to give rise to a differential brain regional distribution; the latter observation rises some concern over the hazard of paraquat as a potential environmental neurotoxin. Indeed, paraquat, administered systemically in rats produces behavioural excitation and brain damage. The brain damage appears to be selective for the pyriform cortex and this does not seem to be strictly related to the high concentrations reached by the herbicide in this area but to the higher vulnerability of this cortical area to the enhanced cholinergic transmission. The recent observation that paraquat, injected into the rat hippocampus, induces the expression of apoptotic neuronal cell death, appears of valuable interest also with a view to paraquat as an useful experimental model in the development of neuroprotective drugs able to block the molecular events which, once activated, are responsible for the induction of neuronal cell death.     

Binding of hirudin to meizothrombin

Prothrombin (coagulation factor II) is the inactive precursor molecule of thrombin (coagulation factor IIa). Proteolytic cleavage of the peptide bond Arg320-Ile321 converts prothrombin into the two-chain thrombin precursor meizothrombin. Meizothrombin hydrolyses peptidyl substrates, but cleavage of fibrinogen is poor. Unfortunately, meizothrombin exhibits a significant autocatalytic activity and thus is not structurally stable in solution. Hirudin, the 65-residue peptide anticoagulant from the salivary gland of the European leech Hirudo medicinalis, is a highly specific and effective thrombin inhibitor. To study the interactions of meizothrombin and hirudin, recombinant prothrombin with active site Asp419 replaced by Asn (D419N-prothrombin) was produced in CHO cells and transformed into D419N-meizothrombin in vitro. D419N-meizothrombin exhibited no proteolytic and autocatalytic activity. D419N-meizothrombin was affinity purified at an immobilized C-terminal hirudin-derived peptide demonstrating the presence and activity of the anion binding exosite. D419N-meizothrombin exhibited binding activity to hirudin immobilized at the solid phase in an ELISA. Incubation of D419N-meizothrombin with hirudin resulted in a significant increase of intrinsic fluorescence. Fluorescence titration of D419N-meizothrombin with hirudin produced a sharp break in the titration curve at the molar equivalence point and a total fluorescence enhancement of 24%. However, the titration curve did not reflect a simple binding mechanism. Incubation of D419N-meizothrombin with fibrinopeptide A and C-terminal hirudin peptide 54-65 did not change fluorescence emission. Trp468 located in the gamma-loop of thrombin was replaced by Phe in the double-mutant D419N/W468F-thrombin. Similar to D419N-thrombin and D419N-meizothrombin, formation of the D419N/W468F-thrombin/hirudin complex resulted a significant increase in intrinsic fluorescence. Apparently, the binding of hirudin induces similar structural changes in both meizothrombin and thrombin. The structural change does not involve the flexible gamma-loop. The results suggest that meizothrombin binds hirudin similar to thrombin.     

The inhibition by substrate analogues of gamma-aminobutyrate aminotransferase from mitochondria of different subcellular fractions of rat brain

Cytoplasmic-mitochondria-enriched and synaptosome-enriched subcellular fractions were prepared from rat brain to study certain kinetic properties of gamma-aminobutyrate: alpha-oxoglutarate aminotransferase (GABA-T) (EC 2.6.1.19) from each of the sources. From this study two differences emerged. Firstly, the cytoplasmic enzyme exhibited an eightfold greater affinity for gamma-aminobutyric acid (GABA) than did the synaptosomal GABA-T; the Km being 6.5 mM and 53 mM, respectively. Secondly, synaptosomal GABA-T is comparatively more susceptible to inhibition by the substrate analogues 2,4-diaminobutyric acid (DABA) and aminooxyacetic acid (AOAA) than is the enzyme from the cytoplasmic mitochondrial fraction. In each case the inhibition was of a competitive nature with respect to GABA. The Ki for the DABA was 13 mM for the cytoplasmic-derived enzyme and 8mM for the synaptosomal enzyme. With AOAA the Ki was 0.1 muM and 0.06 muM for the synaptosomal and cytoplasmic mitochondrial enzyme, respectively. These results provide further evidence that GABA-T from cytoplasmic mitochondria is different in several respects from the enzyme found in synaptosomes.     

Resistance of purified cholera toxin to enzymatic treatment with pancreatic elastase and papain

Treatment of Cholera toxin with pancreatic elastase and papain in vitro showed a high resistance of the toxin molecule to these enzymes, under non-denaturing conditions or in the presence of 2 M urea. These experiments support the hypothesis of a particularly stable molecular structure of the toxin, as an explanation of its activity in the intestinal lumen where the pancreatic proteases are active.     

NAcSDKP analogues resistant to angiotensin-converting enzyme

Two series of analogues of the tetrapeptide NAcSDKP, an inhibitor of hematopoietic stem cell proliferation, were prepared, and their enzymatic stability toward rabbit lung angiotensin-converting enzyme (ACE) was evaluated as well as their capacity to inhibit NAcSDKP hydrolysis by this enzyme. In the first series, each of the peptide bonds has been successively replaced by an aminomethylene bond. In the second one, the C-terminus of the peptide has been modified by decarboxylation or amidation. The results reported here indicate that all of these molecules but one have good stability toward the enzyme but none of the compounds is able to inhibit NAcSDKP hydrolysis by ACE.     

Strategies to target kyotorphin analogues to the brain

The design, synthesis, and pharmacological evaluation of brain-targeted chemical delivery systems (CDS) for a kyotorphin analogue (Tyr-Lys) are described. The brain-targeted compound contains the active peptide in a packaged, disguised form, flanked between the lipophilic cholesteryl ester on the C-terminus and the 1, 4-dihydrotrigonellyl redox targetor, attached to the N-terminus through strategically selected L-amino acid(s) spacer. It was found that for successful brain targeting, the epsilon-amine of Lys needs to be also converted to a lipophilic function. Through sequential enzymatic bioactivation, the Tyr-Lys dipeptide is released in a sustained manner, producing significant and prolonged analgesic activity as demonstrated by the rat tail latency test. An alternate strategy was also employed. Lys was replaced by a redox amino acid pair, Nys+ left and right arrow Nys, the nicotinamide left and right arrow 1,4-dihydronicotinamide analogues of Lys (Nys+ is 2-amino-6-(3-carbamoyl-1-pyridiniumyl)hexanoic acid). The Nys form is lipophilic and facilitates delivery in addition to the C- and N-terminal lipophilic functions. Enzymatic oxidation to Nys+ provides the lock-in, followed by removal of the lipophilic groups, releasing Tyr-Nys+ from the brain-targeted analogue (BTRA). Nys+ was shown to be an effective substitution for Arg or Lys. The activities of the CDS and BTRA, respectively, were antagonized by naloxone, supporting the designed brain-targeted processes. The most potent compound is the two-proline spacer containing CDS (CDS-PP), followed by the BTRA.     

Sequential unfolding of papain in molten globule state

Papain exhibits the characteristics of molten globule under acidic conditions as seen by circular dichroism, fluorescence and ANS binding. Between pH 2.0-2.5 the protein exhibits substantial secondary structure as indicated by far-UV CD spectrum but loses the persistent tertiary interactions of the native state. Enhanced binding of ANS to the state at pH 2.0 in relation to the native and unfolded states at neutral pH indicates a considerable exposure of aromatic side chains. Temperature and guanidine hydrochloride induced unfolding of papain in this state is noncooperative and the transition curves are biphasic in nature. As papain molecule consists of two domains, the results suggest that the domains unfold independently and sequentially.     

Binding of phenol and analogues to alanine complexes of tyrosine phenol-lyase from Citrobacter freundii: implications for the mechanisms of alpha,beta-elimination and alanine racemization

We have examined the interaction of Citrobacter freundii tyrosine phenol-lyase with both L- and D-alanine. This enzyme catalyzes the racemization of alanine as a side reaction, in addition to the physiological beta-elimination of L-tyrosine to give phenol and ammonium pyruvate. The steady-state kinetic parameters for alanine racemization, kcat and Km, for D-alanine are 0.008 S-1 and 32 mM, respectively, while those for L-alanine are 0.03 S-1 and 11 mM. Incubation of tyrosine phenol-lyase with either L- or D-alanine forms a quinonoid complex that exhibits a strong peak at 500 nm. The presence of K+ increases the intensity of the 500-nm absorption with L-alanine, but decreases the intensity of the peak with D-alanine. Rate constants for the formation of these quinonoid intermediates and the effects of phenol and analogues on the reaction with either L- or D-alanine have been studied by rapid-scanning and single-wavelength stopped-flow spectrophotometry. Phenol binds to all the intermediates of tyrosine phenol-lyase with L- and D-alanine, but most strongly to the external aldimine complex, resulting in a decrease in the absorbance at 500 nm at equilibrium. Pyridine N-oxide binds selectively to the quinonoid complex of alanine, and thus causes an increase in the absorbance at 500 nm at equilibrium. 4-Hydroxypyridine causes a decrease in absorbance at 500 nm during the fast phase, but an increase in absorbance at 502 nm in a subsequent slow relaxation.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of gamma-aminobutyric acidA receptors to tubulin

Tubulin cofractionated with gamma-aminobutyric acidA (GABAA) receptors upon affinity chromatography on Affigel 15 coupled to the benzodiazepine Ro 7-1986, and this cofractionation was not due to unspecific adsorption of tubulin to the column material. In addition, GABAA receptors not only bound to microtubules but also coassembled with added tubulin through three cycles of microtubule polymerization and depolymerization. These data indicate that GABAA receptors may be associated with the microtubule cytoskeleton in the brain. In an experiment designed to detect a protein that possibly could form a bridge between tubulin and the GABAA receptor, only a single protein band containing tubulin could be identified that was able to bind polymerized tubulin after sodium dodecyl sulfate-gel electrophoresis of affinity-purified GABAA receptors. These results are discussed with respect to a possible mechanism of association between GABAA receptors and microtubules.     

N-tosyl-L-phenylalanine chloromethyl ketone, a serine protease inhibitor, identifies glutamate 398 at the coenzyme-binding site of human aldehyde dehydrogenase. Evidence for a second "naked anion" at the active site

Human aldehyde dehydrogenase isozymes were inactivated by N-tosyl-L-phenylalanine chloromethyl ketone (TPCK), an inhibitor of chymotrypsin. The inactivation was a first-order process that followed saturation kinetics. NAD and chloral when used together protected against inactivation. In steady-state kinetics, TPCK produced only slope effects versus varied NAD, both slope and intercept effects versus varied glycolaldehyde were produced, indicating that TPCK reacted with the same enzyme form with which NAD reacted. Ki values from steady-state kinetics and saturation kinetics were comparable. Use of [3H]-labeled TPCK showed that inactivation was associated with the incorporation of two molecules of TPCK per molecule of enzyme. The label incorporation occurred into a single tryptic peptide and also into a single chymotryptic peptide of the E1 isozyme. Purification of labeled peptides, followed by sequencing, demonstrated that E398 of aldehyde dehydrogenase was labeled. Reaction of a haloketone, TPCK, with a carboxyl group of E398 indicates that E398 occurs as a "naked anion" within the molecule. This paper constitutes identification of the second (after E268) "naked anion" at the active site of aldehyde dehydrogenase.     

Synthesis of amidrazones using an engineered papain nitrile hydratase

Diadenosine polyphosphates present at the cytosol can be transported to secretory granules allowing their exocytotic release. Extracellularly, they can act through specific metabotropic or ionotropic receptors, or as analogues of P2X and P2Y nucleotide receptors. The specific ionotropic receptor P4 is present in synaptic terminals, and modulated by protein kinases (PK) A and C and protein phosphatases. Activation of PKA or PKC, directly or through membrane receptors, results in a decrease of affinity or in reduction of the Ca2+ transient respectively. Adenosine and ATP, both products of the extracellular destruction of diadenosine polyphosphates, acting through A1 or P2Y receptors respectively, are important physiological modulators at the P4 receptor.     

Binding of naturally occurring anti-DNA antibodies to estradiol

Binding of naturally occurring human anti-DNA autoantibodies to beta-estradiol and native DNA has been investigated. Sera from seven SLE patients were tested for their reactivity with beta-estradiol and native calf thymus DNA. Solid Phase enzyme immunoassay was performed using both colorigenic and fluorogenic substrates. The results show enhanced binding of human anti-DNA autoantibodies to beta-estradiol as compared to native DNA. This was further substantiated by increased binding of affinity purified SLE IgG to beta-estradiol. These results suggest a role for female sex hormone in the etiopathogenesis of SLE and are significant vis-a-vis female predominance of SLE.