Unreacted Enzyme Research Articles

Investigating the position of the hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis and inhibitor binding

In an effort to examine the relative position of a hairpin loop in New Delhi metallo-β-lactamase, NDM-1, during catalysis, rapid freeze quench double electron electron resonance (RFQ-DEER) spectroscopy was used. A doubly-labeled mutant of NDM-1, which had one spin label on the invariant loop at position 69 and another label at position 235, was prepared and characterized. The reaction of the doubly spin labeled mutant with chromacef was freeze quenched at 500μs and 10ms. DEER results showed that the average distance between labels decreased by 4Å in the 500μs quenched sample and by 2Å in the 10ms quenched sample, as compared to the distance in the unreacted enzyme, although the peaks corresponding to distance distributions were very broad. DEER spectra with the doubly spin labeled enzyme with two inhibitors showed that the distance between the loop residue at position 69 and the spin label at position 235 does not change upon inhibitor binding. This study suggests that the hairpin loop in NDM-1 moves over the metal ion during the catalysis and then moves back to its original position after hydrolysis, which is consistent with a previous hypothesis based on NMR solution studies on a related metallo-β-lactamase. This study also demonstrates that this loop motion occurs in the millisecond time domain.

Mass spectrometry of UV-cross-linked protein-nucleic acid complexes: identification of amino acid residues in the single-stranded DNA-binding domain of human replication protein A.

Photochemical cross-linking of human replication protein A (hRPA) to oligonucleotide dT30 was performed to enable identification of amino acid sequences that reside in the DNA-binding domain. A nucleoprotein complex, with a 1:1 protein/DNA stoichiometry, was separated from unreacted enzyme and oligonucleotide by SDS-polyacrylamide gel electrophoresis and subjected to in-gel digestion with trypsin. Three cross-linked tryptic peptides (nucleopeptides) of hRPA70xdT30 (T43, T28/29, and a truncated T24/25) were isolated. Combined mass spectrometric and C-terminal proteolysis experiments showed that at least one amino acid in the segment 235-ATAFNE-240 (located in T24/25), at least one out of the two residues sequence 269-FT-270 (located in T28/29), and at least one from the sequence 383-VSDF-386 (located in T43) were involved in cross-linking. These peptides contained aromatic residues (F238, F269, and F386 respectively) that can form base-stacking interactions with the DNA and were, therefore, most likely to be involved in cross-linking. The results obtained in this study demonstrate that a combination of exhaustive proteolysis and MALDI TOF MS can localize the sites of DNA binding to very short sequences of amino acids. Data so acquired can confirm or amend information obtained from site-directed mutagenesis and X-ray crystallography.

Two Sites of Interaction of Anions with Cytochrome a in Oxidized Bovine Cytochrome c Oxidase

An interaction between cytochrome a in oxidized cytochrome c oxidase (CcO) and anions has been characterized by EPR spectroscopy. Those anions that affect the EPR g = 3 signal of cytochrome a can be divided into two groups. One group consists of halides (Cl-, Br-, and I-) and induces an upfield shift of the g = 3 signal. Nitrogen-containing anions (CN-, NO2-, N3-, NO3-) are in the second group and shift the g = 3 signal downfield. The shifts in the EPR spectrum of CcO are unrelated to ligand binding to the binuclear center. The binding properties of one representative from each group, azide and chloride, were characterized in detail. The dependence of the shift on chloride concentration is consistent with a single binding site in the isolated oxidized enzyme with a Kd of approximately 3 mm. In mitochondria, the apparent Kd was found to be about four times larger than that of the isolated enzyme. The data indicate it is the chloride anion that is bound to CcO, and there is a hydrophilic size-selective access channel to this site from the cytosolic side of the mitochondrial membrane. An observed competition between azide and chloride is interpreted by azide binding to three sites: two that are apparent in the x-ray structure plus the chloride-binding site. It is suggested that either Mg2+ or Arg-438/Arg-439 is the chloride-binding site, and a mechanism for the ligand-induced shift of the g = 3 signal is proposed.

Determination of trypsin, chymotrypsin and kallikrein in porcine pancreas extracts by capillary zone electrophoresis

A new method for the direct detection of trypsin, chymotrypsin A and kallikrein from porcine pancreas by capillary zone electrophoresis is proposed. Optimum conditions for quantitative determination were investigated which also allow the unequivocal distinction of trypsin and chymotrypsin according to porcine or bovine source. For each enzyme good linearity and precision were obtained, with a detection limit of 5.0 μg mL−1. In addition, the formation of the complexes between trypsin and basic pancreatic trypsin inhibitor, as well as that between kallikrein and the same inhibitor, have been investigated. Such complexes can be clearly distinguished either from the unreacted enzyme or the excess inhibitor and their use for analytical purposes has been evidenced. The method is rapid and sensitive and has been applied to determine enzymes in several porcine pancreas extracts. The levels of the active compounds in various kinds of commercial preparations were easily determined with relative standard deviations ranging between 3.3 and 4.9%, and recoveries from 95.8% to 102.4%.

Mechanism of acylphosphatase inactivation by Woodward's reagent K.

The organ common-type (CT) isoenzyme of acylphosphatase is inactivated by Woodward's reagent K (WRK) (N-ethyl-5-phenylisoxazolium-3'-sulphonate) at pH6.0. The inactivation reaction follows apparent pseudo first-order kinetics. The dependence of the reciprocal of the pseudo first-order kinetic constant (kobs) on the reciprocal WRK concentration reveals saturation kinetics, suggesting that the WRK forms a reversible complex with the enzyme before causing inactivation. Competitive inhibitors, such as inorganic phosphate and ATP, protect the enzyme from WRK inactivation, suggesting that this reagent acts at or near to the enzyme active site. The reagent-enzyme adduct, which elicits a strong absorption band with lambdamax at 346 nm, was separated from unreacted enzyme by reverse phase HPLC and the modified protein was cleaved with endoproteinase Glu-C to produce fragments. The HPLC fractionation gave two reagent-labelled peptides (peak 1 and peak 2) that were analysed by ion-spray MS and sequenced. The former is VFFRKHTQAE (residues 20-29 of human CT acylphosphatase) and the latter IFGKVQGVFFRKHTQAE (residues 13-29). MS demonstrated that both peptides are WRK adducts. A fragment ion with m/z of 1171, which is present in the mass spectrum of peak 1, has been identified as a WRK adduct of the peptide fragment 20-26. The lambdamax at 346 nm of WRK adduct suggests that the modified residue is His-25. Five recombinant enzymes mutated in residues included in the 20-29 polypeptide stretch have been produced. Analysis of their reactivities with WRK demonstrates that His-25 is the molecular target of the reagent as its modification causes the inactivation of the enzyme. Since both His-25-->Gln and His-25-->Phe mutants maintain high catalytic activity, we suggest that the observed enzyme inactivation is caused by the reagent (covalently bound to His-25), which shields the active site.

Activation of methotrexate-.alpha.-alanine by carboxypeptidase A monoclonal antibody conjugate

Carboxypeptidase A (CP-A) and monoclonal antibody KS1/4 directed against an antigen on human lung adenocarcinoma cells (UCLA-P3) were derivatized by treatment with succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate and N-succinimidyl 3-(2-pyridyldithio)propionate, respectively. The derivatized proteins were reacted to produce thioether-linked enzyme-antibody conjugates. Sequential HPLC size-exclusion and DEAE chromatography separated the conjugate preparation from unreacted enzyme and antibody. On the basis of SDS-PAGE analysis and measurement of catalytic activity, the preparation contained approximately equal amounts of 1:1 and 2:1 (enzyme:antibody) conjugates; binding activity of the conjugate (1.8 x 10(5) molecules/cell) was similar to that of unreacted antibody. In vitro cytotoxicity studies with UCLA-P3 cells demonstrated the ability of cell-bound conjugate to convert the prodrug methotrexate-alpha-alanine (MTX-Ala) to methotrexate (MTX). In the absence of conjugate, ID50 values for MTX-Ala and MTX were 8.9 x 10(-6) and 5.2 x 10(-8) M, respectively. ID50 for the prodrug improved to 1.5 x 10(-6) M with cells containing bound conjugate. This potentiation of MTX-Ala cytotoxicity by conjugate-bound CP-A, which was at least 30-fold greater than that produced by a comparable amount of free enzyme, is attributed to enhanced effectiveness of MTX generated at the cell surface as opposed to the surrounding medium. Examination of the time course of cytotoxicity over a 96-h period showed that the conjugate-prodrug combination (at 2.5 x 10(-6) M) was nearly as effective as MTX in preventing cell replication. These results demonstrate the chemotherapeutic potential of carboxypeptidase-monoclonal antibody conjugates used in conjunction with MTX peptide prodrugs.

Identification of the essential cysteine residue of NADH-cytochrome b5 reductase.

The soluble catalytic domain of NADH-cytochrome b5 reductase was radiolabeled with [14C]N-ethylmaleimide. Reaction for a limited time resulted in incorporation of 0.41 eq of N-ethylmaleimide and loss of 36% of the enzyme activity. Chromatography on a 5'-ADP affinity column separated the reductase which was modified with N-ethylmaleimide from the unreacted enzyme; the isolated derivative constituted 37% of the total material, was completely inactivated, and contained 1.00 eq of N-ethylmaleimide. Cyanogen bromide cleavage of the derivative demonstrated that radioactivity was limited to a single peptide which contained both Cys-283 and Cys-297. Tryptic hydrolysis of this cyanogen bromide peptide showed that the radioactivity was associated with Cys-283. Automated sequenator analysis confirmed that Cys-283 was the radiolabeled residue. These data demonstrate unambiguously that Cys-283 provides the essential thiol group of cytochrome b5 reductase.

Distribution of reaction intermediates on chicken liver fatty acid synthase.

The distributions of covalent intermediates in the reaction cycle catalyzed by chicken liver fatty acid synthase were studied. In isotope-trapping experiments, 30% of [1-14C] acetyl-enzyme and 6.7% of [2-14C]malonyl-enzyme are converted to long-chain fatty acids, indicating the initiation reaction is partially random. The 3-hydroxybutyryl intermediate is located on the tryptic peptide which contains the 4'-phosphopantetheine (greater than 90%), while the C4-C18 saturated intermediates are distributed both on this peptide and on the peptide that contains the active cysteine. The ratio of intermediates on the two peptides is about unity for chain lengths less than C14, but the amount on the active cysteine progressively decreases for chain lengths of C14, C16, and C18 with trypsinized enzyme. The distributions of carbon chain lengths for the saturated or 3-keto intermediates when acetoacetyl-labeled trypsinized enzyme is incubated with limiting malonyl coenzyme A or NADPH, respectively, show large fractions both of unreacted enzyme and of C16 or longer intermediates. A detailed analysis suggests that the initial condensation and reduction steps are slower than the analogous reactions with longer chain length intermediates. The 3-keto intermediate comprises over 70% of each chain length intermediate detected when NADPH is the limiting substrate, indicating the reduction of the 3-keto intermediates is at least 2 times slower than the reduction of the unsaturated intermediates.

Adducts of Co(II) and Cu(II) Bovine carbonic anhydrase with bidentate ligands

The Zn(II) ion of bovine carbonic anhydrase can be substituted with Co(II), a useful spectroscopic probe, without loss of enzymic activity. The Co(II)-enzyme binds bidentate anionic ligands such as oxalate [1], 2-pyridinecarboxylate [2] and N,N-diethyldithiocarbamate [3], giving pentacoordinate metal derivatives. The ligand displaces the water molecule bound to the metal in the unreacted enzyme, causing a decrease in the 1H NMR relaxation rate to about the same value as in the native diamagnetic Zn(II)-enzyme. The Cu(II)-substituted bovine carbonic anhydrase has no catalytic activity, but is able to bind the same type of ligands as the Co(II)-enzyme. The 1H NMR relaxation rate of the Cu(II)-enzyme, which is almost unaffected by binding of monodentate anions [4], is decreased by binding of the bidentate ones [1, 3, 4]. The substantial residual relaxivity, about 20% that of the unreacted enzyme, may indicate that water coordination is retained in these adducts as it is in the case of monodentate anions, the decrease being related to a different geometry, involving a longer CuO distance. EPR spectra of the adducts with bidentate ligands are compatible with a hexacoordinate tetragonal geometry. The spectroscopic properties of the bicarbonato-derivatives of both Co(II) [1] and Cu(II) [5] enzymes suggest that bicarbonate, the real enzyme substrate, does behave as a bidentate ligand.

Purification of hapten-enzyme conjugates for enzymoimmunoassay

Chromatography on hydroxyapatite, including a potassium phosphate lineargradient elution, was applied to the purification of hapten-enzyme conjugates to be used as immunoenzymatic tracers (namely, progesterone, phenobarbital, diphenylhydantoin coupled to glucose-6-phosphate dehydrogenase). The method effectively removed unreacted enzyme and separated the conjugate classes according to the number of substitutions between enzyme linking groups and hapten derivatives. The adequacy of the Chromatographie procedures in improving the analytical performance of the enzymatic tracer appears to depend on direct interdependence between the degree of substitution and the immunological properties of the conjugates.

8] Immobilization of enzymes on neutral and ionic carriers

This chapter discusses the immobilization of enzymes on neutral and ionic carriers. The immobilization of enzymes by covalent binding on suitable water insoluble polymeric carriers allows manifold variations. The carriers to be effective in the immobilization of enzymes are carriers with anionic matrices obtained from nitrated copolymers of methacrylic acid with methacrylic acid-3-fluoroanilide or fluorostyrenes, and copolymers of methacrylic acid and acrylic acid with 3- or 4-isothiocyanatostyrene. With the reactive carriers, alcohol dehydrogenase, cellulase, chymotrypsin, diastase, glucose oxidase, invertase, papain, alkaline phosphatase, and urease were immobilized with varied success. The binding procedures and investigations with immobilized papain are presented in the chapter as an example. Most of the products show good storage stability in aqueous solution or in the freeze-dried state at 4°. An assay is presented for determination of bound enzyme. Differential determination of the protein content in the enzyme solution before the coupling reaction and the amount of unreacted enzyme after the reaction in the used enzyme and washing solutions (500 ml) is employed using Folin reagent. A new standard calibration plot is made for every measurement.

Affinity Labeling of the Active Site of Staphylococcal Nuclease: REACTIONS WITH BROMOACETYLATED SUBSTRATE ANALOGUES

Abstract The reaction of staphylococcal nuclease with stoichiometric amounts of 3'-(N-14C-bromoacetyl-p-aminophenylphosphoryl)-deoxythymidine 5'-phosphate, an alkylating reagent that is a derivative of a strong competitive inhibitor, deoxythymidine 3',5'-diphosphate, results in inactivation of enzymatic activity. No reaction occurs in the presence of the competitive inhibitor, or when Ca++, which is required for binding of substrates and inhibitors, is omitted from the reaction mixture. The modified enzyme derivative was readily purified and separated from unreacted enzyme since it did not adsorb to a column containing Sepharose to which the competitive inhibitor, deoxythymidine 3'-p-aminophenylphosphoryl 5'-phosphate, was covalently bound. The radioactivity of this protein derivative was accounted for by N-carboxymethyllysine 48 (36%) N-carboxymethyllysine 49 (40%), and O-carboxymethyltyrosine 115 (15%) in the primary structure of the protein. Only one of these residues is alkylated in a given protein molecule. These residues are presumed to be located in the region of the tertiary structure of the enzyme which affords specific affinity for substrates and inhibitors, but which does not determine the hydrolytic rate constants. Staphylococcal nuclease is also inactivated, stoichiometrically, in the presence of Ca++, by the 14C-bromoacetyl derivative of the poorly hydrolyzed substrate, deoxythymidine 5'-p-aminophenylphosphate. This reagent selectively alkylates tyrosine 85, which is thought to occupy a position near the hydrolytic portion of the active site. The reaction occurs either by alkylation followed by hydrolysis, or by both processes occurring in a concerted fashion, possibly through the initial formation of a phosphorylated enzyme-substrate intermediate. The enzyme derivative modified by alkylation at tyrosine 85 is adsorbed to the specific nucleaseinhibitor Sepharose column, indicating that it possesses residual binding function. The derivative is catalytically active, although its affinity for DNA and synthetic substrates is severely depressed. Reaction of staphylococcal nuclease with the bromoacetyl derivative of deoxythymidine 3'-p-aminophenylphosphate, a weak inhibitor of the enzyme, results in considerable nonspecific and random alkylation. Reaction nevertheless occurs selectively with a residue important for catalysis (lysine 24). Although the amount of this specific reaction is small compared to the total reaction with the protein, as judged by incorporation of 14C, no other single residue appears to be as highly labeled.