The interaction of acetate and formate with cobalt carbonic anhydrase. An NMR study.

The interaction of formate and acetate ions with cobalt-substituted carbonic anhydrase (CA) has been investigated through 13C-NMR and one-dimensional and two-dimensional 1H-NMR spectroscopy. 13C data on formate are consistent with a regularly coordinated ligand, as previously proposed for the acetate anion [Bertini, I., Luchinat, C. & Scozzafava, A. (1977) J. Chem. Soc. Dalton Trans., 1962-1965]. 1H-NOE experiments on both anions give evidence of through-space interactions between ligand protons and protein protons. The latter are assigned to specific residues in the active cavity through nuclear Overhauser effect spectroscopy (NOESY) experiments. The 13C-derived and 1H-derived constrains allow reliable docking of these ligands in the active-site cavity. The resulting geometries are similar to one another and consistent with five-coordinated structures around the metal ion, as previously proposed from electronic spectroscopy [Bertini, I., Canti, G., Luchinat, C. & Scozzafava, A. (1978) J. Am. Chem. Soc. 100, 4873-4877]. The results are discussed in light of the current debate on anion binding to metal ions in carbonic anhydrase [Lindahl, M., Svensson, A. & Liljas, A. (1992) Proteins, in the press]; Bertini, I., Luchinat, C., Pierattelli, R. & Vila, A. J. (1992) Inorg. Chem., in the press; Banci, L. & Merz, K. (1992) unpublished results] and, in particular, of the proposed long Zn-O distance found in the recent X-ray results on the formate adduct [Hakanson, K., Carlsson, M., Svensson, A. & Liljas, A. (1992) J. Mol. Biol., in the press].     

Point mutations at the type I Cu ligands, Cys457 and Met467, and at the putative proton donor, Asp105, in Myrothecium verrucaria bilirubin oxidase and reactions with dioxygen

The type I Cu site in the Cys457Ser mutant of Myrothecium verrucaria bilirubin oxidase was vacant, but the trinuclear center composed of a type II Cu and a pair of type III Cu's was fully occupied by three Cu ions. Cys457Ser could react with dioxygen, affording reaction intermediate I with absorption maxima at 340, 470, and 675 nm. This intermediate corresponds to that obtained from laccase, whose type I Cu is cupric and type II and III Cu's are cuprous [Zoppellaro, G., Sakurai, T., and Huang, H. (2001) J. Biochem. 129, 949-953] or whose type I Cu is substituted with Hg [Palmer, A. E., Lee, S. K., and Solomon, E. I. (2001) J. Am. Chem. Soc. 123, 6591-6599]. Another type I Cu mutant, Met467Gln, with modified spectroscopic properties and redox potential, afforded reaction intermediate II with absorption maxima at 355 and 450 nm. This intermediate corresponds to that obtained during the reaction of laccase [Sundaram, U. M., Zhang, H. H., Hedman, B., Hodgson, K. O., and Solomon, E. I. (1997) J. Am. Chem. Soc. 119, 12525-12540; Huang, H., Zoppellaro, G., and Sakurai, T. (1999) J. Biol. Chem. 274, 32718-32724]. According to a three-dimensional model of bilirubin oxidase, Asp105 is positioned near the trinuclear center. Asp105Glu and Asp105Ala exhibited 46 and 7.5% bilirubin oxidase activity compared to the wild-type enzyme, respectively, indicating that Asp105 conserved in all multi-copper oxidases donates a proton to reaction intermediates I and II. In addition, this amino acid might be involved in the formation of the trinuclear center and in the binding of dioxygen based on the difficulties in incorporating four Cu ions in Asp105Ala and Asp105Asn and their reactions with dioxygen.     

Inhibition of serine proteases by peptidyl fluoromethyl ketones

We have synthesized peptidyl fluoromethyl ketones that are specific inhibitors of the serine proteases alpha-chymotrypsin and porcine pancreatic elastase. By analogy with the corresponding aldehydes it is assumed that the fluoromethyl ketones react with the gamma-OH group of the active site serine to form a stable hemiacetal [Lowe, G., & Nurse, D. (1977) J. Chem. Soc., Chem. Commun., 815; Chen, R., Gorenstein, D.G., Kennedy, W.P., Lowe, G., Nurse, D., & Schultz, R.M. (1979) Biochemistry 18, 921; Shah, D.O., Lai, K., & Gorenstein, D.G. (1984) J. Am. Chem. Soc. 106, 4272]. 19F NMR studies of the chymotrypsin-bound trifluoromethyl ketone inhibitors Ac-Leu-ambo-Phe-CF3 and Ac-ambo-Phe-CF3 clearly indicate that the carbonyl carbon is tetrahedral at the active site of the enzyme. The inhibitor is bound as either the stable hydrate or the hemiacetal, involving the active site serine. The effect of varying the number of amino acid residues in the peptidyl portion of the inhibitor and the number of fluorines in the fluoromethyl ketone moiety is examined. In the series of trifluoromethyl ketone elastase inhibitors, the lowering of Ki concomitant with the change from a dipeptide analogue to a tetrapeptide analogue (Ac-Pro-ambo-Ala-CF3, Ki = 3 X 10(-3) M; Ac-Ala-Ala-Pro-ambo-Ala-CF3, Ki = 0.34 X 10(-6) M) correlates well with the variation in V/K for hydrolysis of the corresponding amide substrates. This trend is indicative of the inhibitors acting as transition-state analogues [Bartlett, P.A., & Marlowe, C.K. (1983) Biochemistry 22, 4618; Thompson, R.C. (1973) Biochemistry 12, 47].(ABSTRACT TRUNCATED AT 250 WORDS)     

Competition between nucleoside diphosphates and triphosphates at the catalytic and allosteric sites of phosphorylase kinase

The interactions of nucleotides at the allosteric and catalytic sites of phosphorylase kinase were examined. Binding of nucleoside triphosphates at the nucleoside diphosphate allosteric activation site inhibited enzymatic activity; this was observed with either ATP or GTP. Increasing concentrations of ADP caused a biphasic response: low concentrations activated and higher concentrations inhibited. Inhibition was due to the binding of ADP at the catalytic site, as opposed to an allosteric inhibitory site. GDP activated at low concentrations, but did not inhibit even at relatively high concentrations, and is therefore a specific probe for the allosteric site. Maximal activity of the nonactivated holoenzyme at pH 6.8 is achieved at an optimal ratio of ATP to ADP, such that the inhibitory actions of ATP at the allosteric site and of ADP at the catalytic site are balanced. Various potential molecular mechanisms to explain the allosteric activation by ADP were examined and ruled out, thus strengthening our previous conclusion that the activation is predominantly caused by a conformational transition in the beta subunits directly induced by the binding of ADP (Cheng, A., Fitzgerald, T. J., and Carlson, G. M. (1985) J. Biol. Chem. 260, 2535-2542; Trempe, M. R., and Carlson, G. M. (1987) J. Biol. Chem. 262, 4333-4340; Cheng, A., Fitzgerald, T. J., Bhatnager, D., Roskoski, R., Jr., and Carlson, G. M. (1988) J. Biol. Chem. 263, 5534-5542). The catalytic site exhibited high stereospecificity for inhibition by the Rp and Sp epimers of adenosine 5'-O-(1-thiodiphosphate), with the Rp epimer (Ki = 0.5 microM) being 136-fold more effective than its Sp counterpart. This can readily explain the inability of the Rp epimer to be an effective allosteric activator.     

Analysis of the high- and low-spin Soret bands of horse-heart metmyoglobin complexes

Interest in the thermodynamics of the iron-binding site in hemoproteins has increased in recent years due to refinements in x-ray crystallographic studies of hemoproteins [see Deathage, J. F., Lee, R. S., Anderson, C. M. & Moffat, K. (1976) J. Mol. Biol. 104 , 687–706; Heidner, E. J., Ladner, R. C. & Perutz, M. F. (1976) J. Mol. Biol. 104 , 707–722; Deathage, J. F., Lee, R. S. & Moffat, K. (1976) J. Mol. Biol. 104 , 723–728; Ladner, R. C., Heidner, E. J. & Perutz, M. F. (1976) J. Mol. Biol. 114 , 385–414; Fermi, G. & Perutz, M. F. (1977) J. Mol. Biol. 114 , 421–431; Takano, T. (1977) J. Mol. Biol. 110 , 537–568 and 569–589], the synthesis and x-ray analysis of model heme compounds [see Scheidt, W. R. (1977) Acc. Chem. Res. 10 , 339–345; Kastner, M. E., Scheidt, W. R., Mashino, T. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 666–667; Mashiko, T., Kastner, M. E., Spartalian, K., Scheidt, W. R. & Reed, C. A. (1978) J. Am. Chem. Soc. 100 , 6354–6362; Hill, H. A. O., Skite, P. P., Buchler, J. W., Luchr, H., Tonn, M., Gregson, A. K. & Pellizer, G. (1979) Chem. Commun. 4 , 151–152; and Scheidt, W. R., Cohen, I. A. & Kastner, M. E. (1979) Biochemistry 18 , 3546–3556], and the numerous data on heme–protein interactions that account for the differences observed in ligand binding between the various species of animals. Numerous probes have been used and provide information about the structure and thermodynamics of the binding site, but no single probe can provide the complete picture [see Iizuka, T. & Yonetani, T. (1970) Adv. Biophys. 1 , 157–182; Smith, D. W. & Williams, R. J. P. (1970) Struct. Bond. 7 , 1–45; and Spiro, T. G. (1975) Biochim. Biophys. Acta 416 , 169–189].     

Biosynthesis of lasalocid A: biochemical mechanism for assembly of the carbon framework

Labeling experiments on the biosynthesis of the polyether antibiotic lasalocid A (1) using carboxylic acid precursors bearing 13C, 2H, and 3H labels at various positions established the following: (1) 2H or 3H at C-2 of propionate or 2H at C-2 of butyrate was partially retained at C-12 and C-14 of 1, respectively. (2) 2H at C-2 of propionate or at C-2 and C-3 of succinate did not label C-10. These and earlier data [Hutchinson, C. R., Sherman, M. M., Vederas, J. C., & Nakashima, T. T. (1981) J. Am. Chem. Soc. 103, 5953; Hutchinson, C. R., Sherman, M. M., McInnes, A. G., Walter, J. A., & Vederas, J. C. (1981) J. Am. Chem. Soc. 103, 5956] are consistent with a hypothesis for the stereochemical control of lasalocid A biosynthesis, whose main tenets are that the configuration of C-12 and C-14 is determined by the stereoselectivity of the carbon chain forming condensation between acyl thio ester and 2-carboxyacyl thio ester intermediates and that the configuration of C-11 and C-15 results from the reduction of 2-keto thio ester intermediates with opposing stereospecificities.     

Laboratory Hints from the Literature

DYES AND THEIR BIOLOGICAL USES Burckhalter, J. H., Jones, E. M., Holcomb, W. F., and Sweet, L. A. N-substituted 2-methoxy-6-chloro-9-aminoacridines. J. Amer. Chem. Soc., 65, 2012. 1943.

Galat, Alexander. New processes for sulfanilamide. Ind. and Eng. Chem., Ind. Ed., 36, 192. 1944.

Kumler, W. D., and Daniels, T. C. The relation between chemical structure and bacteriostatic activity of sulfanilamide type compounds. J. Amer. Chem. Soc., 65, 2190. 1943.

Kwartler, C. E., and Lucas, Philip. The preparation of sutfanil-amidoindazoles. J. Amer. Chem. Soc., 65, 1804. 1943.

Mueller, A. C., and Hamilton, C. S. The synthesis of 1-substituted aminobenzo(f)quinolines. J. Amer. Chem. Soc., 65, 1017. 1943.

Popkin, A. H. Derivatives of biphenylsulfonamides. I. Preparation of p-(o-aminopnenyl)-benzenesulfonamide. J. Amer. Chem. Soc., 65, 2043. 1943.

Popkin, A. H., and Perretta, Gertrude M. Derivatives of biphenyl-sulfonamide. II. Derivatives of p-(o-aminophenyl)-benzenesulfonamide. J. Amer. Chem. Soc., 65, 2046. 1943.

Shreve, R. N., and Bennett, R. B. Studies in azo dyes. I. Preparation and bacteriostatic properties of azo derivatives of 2,6-diaminopvridine. J. Amer. Chem. Soc., 65, 2241. 1943.

Shreve, R. N., and Bennett, R. B. Studies in azo dyes. II. Preparation and bacteriostatic properties of azo derivatives of 8-quinolino. J. Amer. Chem. Soc., 65, 2243. 1943.

Siebenmann, C., and Schnitzer, R. J. Chemotherapeutic study of p-nitrobenzoyl- and related compounds. J. Amer. Chem. Soc., 65, 2127 1943.

ANIMAL MICROTECHNIC Barrett, A. M. A method for staining sections of bone marrow. J. Path & Bact., 56, 133-5. 1944.

Barrett, A. M. On the removal of formaldehyde-produced precipitate from sections. J. Path. & Bact., 56, 135-6. 1944.

Chang, Min-Chueh. Disintegration of epidymal spermatozoa by application of ice to the scrotal testis. J. Exp. Biol., 20, 16-22. 1943.

Ercoli, N., and Lewis, M. N. The age factor in response of bone tissue to alizarin dyes and the mechanism of dye fixation. Anat. Rec., 87, 67-76. 1943.

Hess, Manfred, and Hollander, Franklin. Permanent metachromatic staining of gastric mucus smears. J. Lab. and Clin. Med., 29, 321-3. 1944.

Miller, John A. A new method of staining nervous tissue. Ohio J. of Sci., 44, 31-5. 1944.     

Spectra of intermediates in oxidation and reduction of cytochrome c oxidase

Two kinetic components with distinct difference spectra occur during reduction of cytochrome c oxidase by ruthenium hexamine. They are attributed to reduction of heme a (fast phase) and heme a3 (slow phase) (Scott, R. A., and Gray, H. B. (1980) J. Am. Chem. Soc. 102, 3219-3774). Two spectra seen during oxidation of cytochrome c oxidase by molecular oxygen have also been attributed to oxidation of hemes a3 and a (Greenwood, C., and Gibson, Q. H. (1967) J. Biol. Chem. 242, 1782-1787). We now report that spectra for the reductive and oxidative reactions obtained with the same preparations and the same apparatus under similar conditions are significantly different. The reactions appear to populate different reaction intermediates. Reconstitution into phospholipid vesicles does not affect these two spectra significantly. During turnover, the chief intermediates are those of the reductive pathway (Scott and Gray type intermediates). Reduction of heme a3 occurs approximately 70 times faster after turnover than the reduction of the resting enzyme. This is probably a dramatic "pulsing" effect (Wilson, M. T., Peterson, J., Antonini, E., Brunori, M., Colosimo, A., and Wyman, J. (1981) Proc. Natl. Acad. Sci. U.S.A. 7115-7118).     

Oxidation of deuterated compounds by high specific activity methane monooxygenase from Methylosinus trichosporium. Mechanistic implications

Hydrocarbon oxidations catalyzed by methane monooxygenase purified to high specific activity from the type II methanotroph Methylosinus trichosporium OB3b were compared to the same reactions catalyzed by methane monooxygenase from the type I methanotroph Methylococcus capsulatus Bath and liver microsomal cytochrome P-450. The two methane monooxygenases produced nearly identical product distributions, in accord with physical studies of the enzymes which have shown them to be very similar. The products obtained from the oxidation of a series of deuterated substrates by the M. trichosporium methane monooxygenase were very similar to those reported for the same reaction catalyzed by liver microsomal cytochrome P-450, suggesting that the enzymes use similar mechanisms. However, differences in the product distributions and other aspects of the reactions indicated the mechanisms are not identical. Methane monooxygenase epoxidized propene in D2O and d6-propene in H2O without exchange of substrate protons or deuterons with solvent, in contrast to cytochrome P-450 (Groves, J. T., Avaria-Neisser, G. E., Fish, K. M., Imachi, M., and Kuczkowski, R. L. (1986) J. Am. Chem. Soc. 108, 3837-3838), suggesting that the mechanism of epoxidation of olefins by methane monooxygenase differs at least in part from that of cytochrome P-450. Hydroxylation of alkanes by methane monooxygenase revealed close similarities to hydroxylations by cytochrome P-450. Allylic hydroxylation of 3,3,6,6-d4-cyclohexene occurred with approximately 20% allylic rearrangement in the case of methane monooxygenase, whereas 33% was reported for this reaction catalyzed by cytochrome P-450 (Groves, J. T., and Subramanian, D. V. (1984) J. Am. Chem. Soc. 106, 2177-2181). Similarly, hydroxylation of exo,exo,exo,exo-2,3,5,6-d4-norbornane by methane monooxygenase occurred with epimerization, but to a lesser extent than reported for cytochrome P-450 (Groves, J. T., McClusky, G. A., White, R. E., and Coon, M. J. (1978) Biochem. Biophys. Res. Commun. 81, 154-160). A large intramolecular isotope effect, kH,exo/kD,exo greater than or equal to 5.5, was calculated for this reaction. However, the intermolecular kinetic isotope effect on Vm for methane oxidation was small, suggesting that steps other than C-H bond breakage were rate limiting in the overall enzymatic reaction. Similar isotope effects have been observed for cytochrome P-450. These observations indicate a stepwise mechanism of hydroxylation for methane monooxygenase analogous to that proposed for cytochrome P-450.(ABSTRACT TRUNCATED AT 400 WORDS)     

X-ray absorption spectroscopic studies of the high-spin iron(II) active site of isopenicillin N synthase: evidence for Fe-S interaction in the enzyme-substrate complex.

Isopenicillin N synthase from Cephalosporium acremonium (IPNS; M(r) 38.4K) is an Fe(2+)-requiring enzyme which catalyzes the oxidative conversion of (L-alpha-amino-delta-adipoyl)-L-cysteinyl-D-valine (ACV) to isopenicillin N, with concomitant reduction of O2 to 2H2O. Chemical and spectroscopic data have suggested that catalysis proceeds via an enzyme complex of ACV bound to the iron through its cysteinyl thiolate [Baldwin, J. E., & Abraham, E. P. (1988) Nat. Prod. Rep. 5, 129-145; Chen, V. J., Orville, A. M., Harpel, M. R., Frolik, C. A., Surerus, K. K., Münck, E., & Lipscomb, J. D. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C. A., & Chen, V. J. (1991) Biochemistry 30, 11653-11659]. Here we have employed the technique of Fe K-edge extended X-ray absorption fine structure (EXAFS) to characterize the iron site and to seek direct evidence for or against the formation of an Fe-S interaction upon ACV binding. Our data collected in the absence of substrate and O2 are consistent with the iron center of IPNS being coordinated by only (N,O)-containing ligands in an approximately octahedral arrangement and with an average Fe-(N,O) distance of 2.15 +/- 0.02 A. Upon anaerobic binding of ACV, the iron coordination environment changes considerably, and the associated Fe EXAFS cannot be adequately simulated without incorporating an Fe-S interaction at 2.34 +/- 0.02 A along with four or five Fe-(N,O) interactions at 2.15 +/- 0.02 A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Light-induced charge separation in Rhodopseudomonas viridis reaction centers monitored by Fourier-transform infrared difference spectroscopy: the quinone vibrations.

Static FTIR light-induced difference spectra have been recorded for reaction centers from Rhodopseudomonas viridis in the following charge-separated states: P+QA(-)-PQA, P+QB(-)-PQB, I(-)-I, I-QA(-)-IQA, and I-QA(2-)-IQA. A comparison of the I(-)-I difference spectra with the I-QA(-)-IQA difference spectra reveals new bands which can be assigned to QA- vibrations; these vibrations are also observed in the P+QA(-)-PQA and P+QB(-)-PQB difference spectra. Through an analysis of all of the static difference spectra, the electron-transfer pathway can be monitored in the infrared from the primary donor, P, to the secondary acceptor, QB, via the intermediate acceptor, I, and the primary acceptor, QA. The difference spectra are dominated by absorbance changes of prosthetic groups, with very few identifiable contributions from amino acids and little overall structural change in the protein backbone, involving only one or two residues for the various charge-separated states. Oxidation of the primary donor in the reaction center shows the characteristic absorbance changes of the 9-keto and 10-ester carbonyl groups observed upon oxidation of bacteriochlorophyll b in a non-hydrogen-bonded environment [Ballschmiter, K. H., & Katz, J. J. (1969) J. Am. Chem. Soc. 91, 2661-2677]. Reduction of the quinones in the reaction center yields absorbance changes of the carbonyls observed during reduction of quinones in a hydrogen-bonded environment [Bauscher, M., Nabedryk, E., Bagley, K., Breton, J., & M?ntele, W. (1990) FEBS Lett. 261, 191-195].(ABSTRACT TRUNCATED AT 250 WORDS)     

Phospholipids chiral at phosphorus. Synthesis of chiral phosphatidylcholine and stereochemistry of phospholipase D

Chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphocholines (DPPC) with known configuration were synthesized by N-methylation of chirally labeled 1,2-dipalmitoyl-sn-glycero-3-phosphoethanolamine (DPPE). Transphosphatidylation of (RP)- and (SP)-[18O]DPPC catalyzed by phospholipase D from cabbage gave (RP)- and (SP)-[18O]DPPE, respectively, as indicated by 31P nuclear magnetic resonance (NMR) analysis of [18O]DPPE. Therefore, phospholipase D catalyzes transphosphatidylation with overall retention of configuration at phosphorus. The steric course of hydrolysis of DPPC catalyzed by the same enzyme was elucidated by the following procedures. Hydrolysis of (RP)-[17O, 18O]DPPC by phospholipase D gave 1,2-dipalmitoyl-sn-glycero-3-[ 16O , 17O, 18O]phosphate ( [ 16O , 17O, 18O] DPPA ) with unknown configuration. The latter compound was then converted to 1-[ 16O , 17O, 18O]phospho-(R)-propane-1,2-diol by a procedure involving no P-O bond cleavage [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The configuration of the phosphopropane -1,2-diol was determined as RP by 31P NMR analysis following ring closure and methylation [ Buchwald , S. L., & Knowles, J. R. (1980) J. Am. Chem. Soc. 102, 6601-6603]. The results indicated that hydrolysis of DPPC catalyzed by phospholipase D also proceeds with retention of configuration at phosphorus. Our results therefore support a two-step mechanism involving a phosphatidyl-enzyme intermediate in the reactions catalyzed by phospholipase D from cabbage.     

Stimulation of mitogen-activated protein kinase by G protein-coupled alpha(2)-adrenergic receptors does not require agonist-elicited endocytosis.

Agonist-elicited receptor sequestration is strikingly different for the alpha(2A)- versus alpha(2B)-adrenergic receptor (alpha(2)-AR) subtypes; the alpha(2B)-AR undergoes rapid and extensive disappearance from the HEK 293 cell surface, whereas the alpha(2A)-AR does not (Daunt, D. A., Hurt, C., Hein, L., Kallio, J., Feng, F., and Kobilka, B. K. (1997) Mol. Pharmacol. 51, 711-720; Eason, M. G., and Liggett, S. B. (1992) J. Biol. Chem. 267, 25473-25479). Since recent reports suggest that endocytosis is required for some G protein-coupled receptors to stimulate the mitogen-activated protein (MAP) kinase cascade (Daaka, Y., Luttrell, L. M., Ahn, S., Della Rocca, G. J., Ferguson, S. S., Caron, M. G., and Lefkowitz, R. J. (1998) J. Biol. Chem. 273, 685-688; Luttrell, L. M., Daaka, Y., Della Rocca, G. J., and Lefkowitz, R. J. (1997) J. Biol. Chem. 272, 31648-31656; Ignatova, E. G., Belcheva, M. M., Bohn, L. M., Neuman, M. C., and Coscia, C. J. (1999) J. Neurosci. 19, 56-63), we evaluated the differential ability of these two subtypes to activate MAP kinase. We observed no correlation between subtype-dependent agonist-elicited receptor redistribution and receptor activation of the MAP kinase cascade. Furthermore, incubation of cells with K(+)-depleted medium eliminated alpha(2B)-AR internalization but did not eliminate MAP kinase activation, suggesting that receptor internalization is not a general prerequisite for activation of the MAP kinase cascade via G(i)-coupled receptors. We also noted that neither dominant negative dynamin (K44A) nor concanavalin A treatment dramatically altered MAP kinase activation or receptor redistribution, indicating that these experimental tools do not universally block G protein-coupled receptor internalization.     

Structure of oligosaccharides and the linkage region between keratan sulfate and the core protein on proteoglycans from monkey cornea

Structural analyses were performed on the intact glycopeptides and on the linkage region oligosaccharide-peptides derived from the keratan sulfate proteoglycan from monkey cornea (Nakazawa, K., Newsome, D.A., Nilsson, B., Hascall, V.C., and Hassell, J.R. (1983) J. Biol. Chem. 258, 6051-6055) using trifluoroacetolysis, Smith degradation, chromium trioxide oxidation, and gas-liquid chromatography-mass spectrometry. The following structure was found for the linkage region (formula; see text) The following structures were found for the intact oligosaccharide peptides (formula; see text) and (formula; see text) The structure of the linkage region for keratan sulfate on corneal proteoglycans is clearly derived from a complex type of N-linked glycoprotein oligosaccharide precursor, indicating that only the oligosaccharides that have been processed to the complex type are used as primers for synthesizing keratan sulfate chains. The high mannose oligosaccharide in Formula 3 is an intermediate in the normal pathway for biosynthesis of complex type oligosaccharides. The structure in Formula 2, in which a single Man alpha 1-2 is retained on the Man alpha 1-3 branch while the Man alpha 1-6 branch is unsubstituted, can be an intermediate for an alternate, presumably minor pathway for complex oligosaccharide formation (Kornfeld, S., Gregory, W., and Chapman, A. (1979) J. Biol. Chem. 254, 11649-11654) in certain cases. This structure has not previously been shown to be present on normal glycoproteins.     

CO bond angle changes in photolysis of carboxymyoglobin

Previous studies [Chance, B., Fischetti, B., & Powers, L. (1983) Biochemistry 22, 3820-3829] of the local structure changes around the iron in carboxymyoglobin on photolysis at 4 K revealed that the iron-carbon distance increased approximately 0.05 A but was accompanied by a lengthening of the iron-pyrrole nitrogen bonds of the heme (approximately 0.03 A) that was not as large as that found in the deoxy form. Further analysis of these data together with comparison to model compounds indicates that the Fe-C-O bond angle in carboxymyoglobin is bent (127 +/- 4 degrees), having a structure identical, within the error, with the "pocket" porphyrin model compound FePocPiv(1-MeIm)(CO) [Collman, J. P., Brauman, J. I., Collins, T. J., Iverson, B. L., Lang, G., Pettman, R., Sessler, J. L., & Walters, M. A. (1983) J. Am. Chem Soc. 105, 3038-3052]. On photolysis, this angle decreases by 5-10 degrees. In addition, correlation is observed between the increase in the length of the Fe-C bond and the decrease of the Fe-C-O angle. These results suggest that the rate-limiting step in recombination is the thermal motion of CO in the pocket to achieve an appropriate bonding angle with respect to the iron. These changes constitute the first molecular picture of the photolysis process, as well as the structure of the geminate state, and are important in clarifying nuclear tunneling parameters.     

NMR studies of the active site of isopenicillin N synthase, a non-heme iron(II) enzyme.

The active site structure of isopenicillin N synthase (IPNS) has been previously studied by the use of M?ssbauer, EPR, electronic absorption, and NMR spectroscopies [Chen, V.J., Frolik, C.A., Orville, A.M., Harpel, M.R., Lipscomb, J.D., Surerus, K.K., & Münck, E. (1989) J. Biol. Chem. 264, 21677-21681; Ming, L.-J., Que, L., Jr., Kriauciunas, A., Frolik, C.A., & Chen, V.J. (1990) Inorg. Chem. 26, 1111-1112]. These studies have revealed three coordinated His residues along with three sites for substrate [delta-(L-alpha-aminoadipoyl)-L-cysteinyl-D-valine, ACV], NO, and water binding on the active Fe(II) of IPNS. We report here NMR studies of Fe(II)IPNS and its Co(II)-substituted derivative [Co(II)IPNS]. By the use of NOE techniques on the Co(II)IPNS-ACV complex, we have recognized a -CH2-CH less than spin system at 14.6, 24.3, and 38.6 ppm that is assigned to the alpha and beta protons of a coordinated Asp residue. Corresponding solvent nonexchangeable features are found near 40 ppm in Fe(II)IPNS and the Fe(II)IPNS-ACV complex, but the peaks are too broad for NOE effects to be observed. The binding of NO to the Fe(II) center results in a significant change in the configuration of the metal site: (a) The C beta H2 resonances due to the coordinated Asp residue disappear. The loss of the signal may indicate a change of the carboxylate configuration from syn-like to anti-like or, less likely, its displacement by NO.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of the insoluble PiZ variant of human alpha 1-antitrypsin within the hepatic endoplasmic reticulum does not elevate the steady-state level of grp78/BiP

Greater than 85% of the transport-impaired PiZ variant of human alpha 1-antitrypsin is retained within cells and subsequently degraded within a pre-Golgi nonlysosomal compartment that is apparently separate from the endoplasmic reticulum (ER) (Le, A., Graham, K. S., and Sifers, R. N. (1990) J. Biol. Chem. 265, 14001-14007). Despite this phenomenon, human patients and PiZ-bearing transgenic mice exhibit an accumulation of the undegraded protein as insoluble aggregates within distended cisternae of the hepatic ER (Carlson, J. A., Rogers, B. B., Sifers, R. N., Finegold, M. J., Clift, S. M., DeMayo, F. J., Bullock, D. W., and Woo, S. L. C. (1989) J. Clin. Invest. 83, 1183-1190). Immunoprecipitation of the PiZ variant from pulse-radiolabeled hepatocytes from the transgenic animals has demonstrated that a minute quantity of the newly synthesized mutant protein is apparently resistant to degradation and accumulates gradually within the particulate fraction of the cell. Although the steady-state level of the resident ER protein grp78/BiP is elevated in response to the accumulation of malfolded proteins within that subcellular compartment, this phenomenon is not elicited by the accumulation of the insoluble PiZ variant. These results indicate that neither the accumulation of this malfolded protein within the ER nor even the distention of that subcellular compartment is sufficient to cause the up-regulation of grp78/BiP levels. The interpretation of these results with regard to the factors that regulate the levels of grp78/BiP in the ER is discussed.     

Phospholipids chiral at phosphorus. Absolute configuration of chiral thiophospholipids and stereospecificity of phospholipase D

Separate diastereomers of 1,2-dipalmitoyl-sn-glycero-3- thiophosphoethanolamine ( DPPsE ) were prepared in 97% diastereomeric purity and characterized by 31P, 13C, and 1H nuclear magnetic resonance (NMR). The isomers hydrolyzed by phospholipases A2 and C specifically were designated as isomer B (31P NMR delta 59.13 in CDCl3 + Et3N ) and isomer A (59.29 ppm), respectively, analogous to the isomers B and A of 1,2-dipalmitoyl-sn-glycero-3- thiophosphocholine ( DPPsC ) [ Bruzik , K., Jiang , R.-T., & Tsai, M.-D. (1983) Biochemistry 22, 2478-2486]. Phospholipase D from cabbage was shown to be specific to isomer A of DPPsC in transphosphatidylation . The product DPPsE was shown to be isomer A. The absolute configuration of chiral DPPsE at phosphorus was elucidated by bromine-mediated desulfurization in H2 18O to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphoethanolamine ( [18O]DPPE) followed by 31 P NMR analysis [ Bruzik , K., & Tsai, M.-D. (1984) J. Am. Chem. Soc. 106, 747-754]. The absolute configuration of chiral DPPsC was elucidated by desulfurization in H2 18O mediated by bromine or cyanogen bromide to give chiral 1,2-dipalmitoyl-sn-glycero-3-[18O]phosphocholine ( [18O]DPPC), which was then converted to [18O]DPPE by phospholipase D with retention of configuration [ Bruzik , K., & Tsai, M.-D. (1984) Biochemistry (preceding paper in this issue)]. The results indicate that isomer A of both DPPsE and DPPsC is SP whereas isomer B is RP.