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- ArticleHeckler TG, Day RA.Biochim Biophys Acta. 1983 Jun 29;745(3):292-300.Penicillinase from Bacillus cereus 569/H was purified to homogeneity. Its active site was probed by use of an affinity label generated in situ by the diazotization of 6-aminopenicillanic acid, a catalytically poor substrate for this enzyme. The loss of activity arising during the inactivation is dependent upon pH and the penicillin:sodium nitrite ratio used. Optimal inactivation was obtained at pH 4.7 and reactivation could be prevented if subsequent purification and manipulations were performed at low pH. Inactivation by diazotized 6-aminopenicillanic acid was characterized further by tryptic and chymotryptic digestion of the inactivated enzyme and peptide mapping of the resulting digests. Amino acid analysis of the chymotryptic labeled peptide yielded a composition which corresponds to residues 41-46 (Ala-Phe-Ala-Ser-Thr-Tyr) in the published partial sequence of the enzyme (Thatcher, D. (1975) Biochem. J. 147, 313-326). Further digestion of this chymotryptic peptide with carboxypeptidase A reveals that serine-44 is modified in this affinity labeling procedure. Mass spectral analysis of the modified serine residue and alkali-released label, and comparison with spectra of model compounds indicates that the inactivation occurs with rearrangement of the beta-lactamthiazolidine structure to a dihydrothiazine.
- ArticleRavi A, Balaram P.Biochim Biophys Acta. 1983 Jun 29;745(3):301-9.A synthetic model peptide, (formula; see text) which mimics the active-site disulfide loop of thioredoxin has been prepared. 270 MHz 1H-NMR studies establish that Cys-4 and methylamide NH groups are solvent-shielded, using hydrogen-deuterium exchange, solvent and temperature dependence of chemical shifts and nitroxide radical-induced broadening as diagnostic criteria. Infrared measurements provide supporting evidence for intramolecularly hydrogen-bonded conformations. The related peptide in which Gly-2 is replaced by alpha-aminoisobutyric acid has been shown to adopt a similar backbone conformation based on NMR and CD data. Based on the known stereochemical preferences of alpha-aminoisobutyric acid residues, a consecutive beta-turn conformation involving two intramolecular 4 leads to 1 hydrogen bonds is proposed for both disulfides. Vicinal coupling constants and CD data are discussed with reference to the side-chain conformation of the cysteine residues. Large structural differences have been established between the thioredoxin active-site model disulfide and its acyclic precursor.
- ArticleMawatari K, Matsukawa S, Yoneyama Y.Biochim Biophys Acta. 1983 Jun 29;745(3):219-28.Absorption and circular dichroism (CD) spectra of the Soret band, assigned as a pi-pi transition of the porphyrin pi-electron system, showed a great difference between alpha and beta subunits in the ferric state (alpha +, beta +). The nonequivalence of the spectra between alpha + and beta + subunits partly originates from the difference in the strength of the bond between heme iron and the proximal histidine. The peak positions for absorption and CD spectra of the ferric derivatives associated with F-, H2O, N-3 and CN- of the isolated subunits qualitatively correlate with the spin state of the ferric heme. The Soret absorption spectra obtained by simple addition of those for alpha + and beta + subunits are very similar to those for methemoglobin A (metHb A). On the other hand, the arithmetic means for the Soret CD spectra of alpha + and beta + subunits are different from those for metHb A. These differences were not observed between the Soret CD spectra of alpha 1 beta 1 dimer, which is predominantly present in metHb Hirose (beta 37Trp-Ser), and those of tetrameric metHb A. Therefore, the interaction between alpha 1 and beta 1 subunits to make the alpha 1 beta 1 dimer may strongly affect the CD spectral properties of alpha + and beta + subunits. The effect of the interaction between two homogeneous dimers, alpha 1 beta 1 and alpha 2 beta 2, forming a tetramer, on the Soret CD spectral properties, if any, is very small compared with that between alpha 1 and beta 1 subunits.
- ArticleThompson RE, Morrical SW, Campbell DP, Carper WR.Biochim Biophys Acta. 1983 Jun 29;745(3):279-84.The conformational changes in glucose dehydrogenase are studied as a function of temperature and guanidinium chloride (GdmCl) concentration. The data were analyzed assuming a two-conformer model which gave similar results using either circular dichroism or enzyme activity. The free energy of denaturation was 0.94 kcal/mol from specific activity and 1.64 kcal/mol from circular dichroism measurements. The mid-point of the denaturation curve was 0.65 or 0.63 M GdmCl, as determined by specific activity or circular dichroism, respectively. The transition temperature, 6.4 degrees C, is close to that of a microsomal membrane phase change, a result that is consistent with the fact that glucose dehydrogenase contains lipid materials when isolated with a non-ionic detergent such as Triton X-114. As the temperature increased, the amount of beta-pleated sheet increased, and the alpha-helical content decreased, suggested that glucose dehydrogenase contains a stable core of beta-pleated sheet.
- ArticleStone SR, Morrison JF.Biochim Biophys Acta. 1983 Jun 29;745(3):237-46.Binding theory has been developed for the reaction of an ionizing enzyme with an ionizing ligand. Consideration has been given to the most general scheme in which all possible reactions and interconversions occur as well as to schemes in which certain interactions do not take place. Equations have been derived in terms of the variation of the apparent dissociation constant (Kiapp) as a function of pH. These equations indicate that plots of pKiapp against pH can be wave-, half-bell- or bell-shaped according to the reactions involved. A wave is obtained whenever there is formation of the enzyme-ligand complexes, ionized enzyme . ionized ligand and protonated enzyme . protonated ligand. The additional formation of singly protonated enzyme-ligand complexes does not affect the wave form of the plot, but can influence the shape of the overall curve. The formation of either ionized enzyme . ionized ligand or protonated enzyme . protonated ligand, with or without singly protonated enzyme-ligand species, gives rise to a half-bell-shaped plot. If only singly protonated enzyme-ligand complexes are formed the plots are bell-shaped, but it is not possible to deduce the ionic forms of the reactants that participate in complex formation. Depending on the reaction pathways, true values for the ionization and dissociation constants may or may not be determined.
- ArticleStone SR, Morrison JF.Biochim Biophys Acta. 1983 Jun 29;745(3):247-58.The interaction of dihydrofolate reductase (EC 1.5.1.3) from Escherichia coli with dihydrofolate and folate analogues has been studied by means of binding and spectroscopic experiments. The aim of the investigation was to determine the number and identity of the binary complexes that can form, as well as pKa values for groups on the ligand and enzyme that are involved with complex formation. The results obtained by ultraviolet difference spectroscopy indicate that, when bound to the enzyme, methotrexate and 2,4-diamino-6,7-dimethylpteridine exist in their protonated forms and exhibit pKa values for their N-1 nitrogens of above 10.0. These values are about five pH units higher than those for the compounds in free solution. The binding data suggest that both folate analogues interact with the enzyme to yield a protonated complex which may be formed by reaction of ionized enzyme with protonated ligand and/or protonated enzyme with unprotonated ligand. The protonated complex formed with 2,4-diamino-6,7-dimethylpteridine can undergo further protonation to form a protonated enzyme-protonated ligand complex, while that formed with methotrexate can ionize to give an unprotonated complex. A group on the enzyme with a pKa value of about 6.3 is involved with the interactions. However, the ionization state of this group has little effect on the binding of dihydrofolate to the enzyme. For the formation of an enzyme-dihydrofolate complex it is essential that the N-3/C-4 amide of the pteridine ring of the substrate be in its neutral form. It appears that dihydrofolate is not protonated in the binary complex.
- ArticleKubilus J, Baden HP.Biochim Biophys Acta. 1983 Jun 29;745(3):285-91.The deimination of guanidyl groups of peptides, proteins and other arginine-containing compounds is catalyzed by enzymes found in mammalian brain and epidermis. In cow, the brain and epidermal enzymes differ kinetically and physically, but both may be quantitated by measuring the production of benzoyl citrulline ethyl ester from benzoyl-arginine ethyl ester. The brain enzyme has been purified to apparent homogeneity, as judged by the presence of only one 85,000 dalton band in purified preparations when examined by SDS-polyacrylamide gel electrophoresis. An antibody raised to this band precipitates pure and partially purified brain enzyme but not partially purified epidermal enzyme, using the Ouchterlony technique. The antibody bound to an insoluble matrix removes brain enzyme activity from solution but not epidermal enzyme activity. The Km of the brain enzyme for benzoyl-arginine ethyl ester is about 0.33 mM.
- ArticlePinkwart M, Schneider K, Schlegel HG.Biochim Biophys Acta. 1983 Jun 29;745(3):267-78.The nitrogen-fixing, aerobic hydrogen-oxidizing bacterium Alcaligenes latus forms hydrogenase when growing lithoautotrophically with hydrogen as electron donor and carbon dioxide as sole carbon source or when growing heterotrophically with N2 as sole nitrogen source. The hydrogenase is membrane-bound and relatively oxygen-sensitive. The enzymes formed under both conditions are identical on the basis of the following criteria: molecular mass, mobility in polyacrylamide gel electrophoresis, Km value for hydrogen (methylene blue reduction), stability properties, localization, and cross-reactivity to antibodies raised against the 'autotrophic' hydrogenase. The hydrogenase was solubilized by Triton X-100 and deoxycholate treatment and purified by ammonium sulfate precipitation and chromatography on Phenyl-Sepharose C1-4B, DEAE-Sephacel and Matrix Gel Red A under hydrogen to homogeneity to a specific activity of 113 mumol H2 oxidized/min per mg protein (methylene blue reduction). SDS gel electrophoresis revealed two nonidentical subunits of molecular weights of 67 000 and 34 000, corresponding to a total molecular weight of 101 000. The pure enzyme was able to reduce FAD, FMN, riboflavin, flavodoxin isolated from Megasphaera elsdenii, menadione and horse heart cytochrome c as well as various artificial electron acceptors. The reversibility of the hydrogenase function was demonstrated by H2 evolution from reduced methyl viologen.
- ArticleCraven PA, DeRubertis FR.Biochim Biophys Acta. 1983 Jun 29;745(3):310-21.Guanylate cyclase activity was purified to apparent homogeneity from rat liver (7700-fold) and bovine lung (8600-fold) soluble fractions by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and isoelectric focussing. The purified enzymes did not contain heme and did not respond to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. By contrast, preformed NO-hemoglobin increased enzyme activity 10-12-fold or 60-80-fold when 4 mM MnCl2 or 4 mM MgCl2, respectively, were employed as the metal ion co-factor. Addition of hematin to the enzyme preparations restored responsiveness to NO, nitroprusside or NO-cysteine to levels seen with NO-hemoglobin. Partial purification of guanylate cyclase from the soluble fraction of bovine lung (2400-fold) by ammonium sulfate precipitation, DEAE-cellulose chromatography, agarose gel filtration and high pressure liquid chromatography (HPLC) resulted in a preparation which contained endogenous heme as indicated by absorbance at 436 nm and responded to NO, nitroprusside and NO-cysteine in the absence of added hematin. By contrast, guanylate cyclase purified from the hepatic supernatant by the identical procedure, did not contain detectable absorption due to heme and did not respond or responded poorly to NO, nitroprusside or NO-cysteine in the absence of exogenous hematin. Analogous to hepatic guanylate cyclase purified by isoelectric focussing, the HPLC purified hepatic enzyme was activated 14-fold by NO-hemoglobin in assays which contained 4 mM MnCl2 and 60-fold in assays with 4 mM MgCl2. Further, addition of hematin to the HPLC purified enzyme restored responsiveness to NO, nitroprusside and NO-cysteine to levels seen with NO-hemoglobin. These effects of hematin were specific for hematin and were not mimicked by albumin, sucrose or dithiothreitol. Moreover, the failure to observe stimulation of purified hepatic guanylate cyclase was not explained by a shift in the concentration response relationship between NO and guanylate cyclase activity. Several observations indicated that neither NO-thiol complexes nor [Fe(CN)5NO]-3 were the proximate moieties responsible for activation of guanylate cyclase by nitroprusside and related agents, as has been previously suggested. These results strongly support the proposal that activation of guanylate cyclase by NO and related agents specifically requires formation of an NO-heme complex.
- ArticleDreyer JL, Beinert H, Keana JF, Hankovszky OH, Hideg K, Eaton SS, Eaton GR.Biochim Biophys Acta. 1983 Jun 29;745(3):229-36.It has been reported by Johnson et al. ((1977) Biochem. Biophys. Res. Commun. 74, 384-389) that phenacyl bromide reacts with a single reactive sulfhydryl group of aconitase, abolishing enzyme activity. Substrate or analogs have a protective effect. This group is therefore at the catalytic site of the enzyme. Aconitase is also known to be an Fe-S protein, paramagnetic as obtained on purification (Ruzicka and Beinert (1978) J. Biol. Chem. 253, 2514-2517). We have attempted to obtain information on the location of the Fe-S cluster of aconitase with respect to the catalytically active site by attaching nitroxide-labelled sulfhydryl reagents of the bromoacyl and maleimide type to the sensitive sulfhydryl group. The EPR signals of those spin-labelled sulfhydryl reagents that abolish enzyme activity disappear during reaction with aconitase. EPR spectra at 13 K of the product obtained by reaction of three spin labels (two maleimides and one bromoacyl) with aconitase included a half-field transition at g approximately equal to 4.0 which is characteristic of spin-spin interaction. On the basis of calculations of the dependence of the intensity of the half-field transition on the distance between two interacting unpaired electrons (Eaton and Eaton, (1982) J. Am. Chem. Soc. 104, 5002-5003) the distances between the nitroxide N-O bond and the center of the Fe-S cluster for the three spin labels were calculated to be 10.5, 11 and 13 A. Combined distance and orientation data for the three spin labels indicate that the reactive sulfhydryl group is about 12 A from the center of the Fe-S cluster.