Spectral properties of cobalt carboxypeptidase A. Interaction of the metal atom with anions

At pH greater than 7 the absorption and magnetic circular dichroic spectra of cobalt carboxypeptidase A are insensitive to anions [Latt, S. A., & Vallee, B. L. (1971) Biochemistry 10, 4263-4270], but at pH less than 6 chloride and other anions perturb them in a manner specific for each anion. Lowering of the pH apparently facilitates the entry of an anion into the metal coordination sphere, suggesting that an acidic group normally stabilizes a metal-coordinated water molecule against displacement. The lack of sensitivity to anions at pHs between 7 and 9--when the enzyme is maximally active--and its evident abolition upon protonation of an active-site group are consistent with this interpretation. Selective modification of cobalt carboxypeptidase at Glu-270 using a carbodiimide affinity reagent generates sensitivity to anions at pH 7 very similar to that of the unmodified enzyme at pH approximately 5. This suggests that the group stabilizing the metal-coordinated water is the catalytically essential carboxylate of Glu-270. These and related results provide evidence for a mechanistically important interaction of Glu-270 with a metal-bound water molecule.     

Changes in the coordination geometry of the active-site metal during catalysis of benzylpenicillin hydrolysis by Bacillus cereus beta-lactamase II

Rapid-scanning stopped-flow spectroscopy (425-700 nm) has been used to study spectral changes in cobalt(II)-substituted Bacillus cereus beta-lactamase II during the binding and hydrolysis of benzylpenicillin. The experiments were carried out in aqueous solution over a temperature range of 3-20 degrees C. Three metallointermediates have been characterized by their visible absorption spectra. Two of them have visible absorption spectra identical with the intermediates ES1 and ES2 previously observed at subzero temperatures in a mixed aqueous/organic solvent [Bicknell, R., & Waley, S.G. (1985) Biochemistry 24, 6876-6887]. In addition, the branched kinetic pathway observed with the zinc(II) and cobalt(II) beta-lactamase II at subzero temperatures has been shown to occur with the cobalt(II)-substituted enzyme in aqueous solution at above-zero temperatures; thus, at pH 6.0 and 3 degrees C, the rate and equilibrium constants are readily determined for the reaction scheme: (Formula: see text). A third transient intermediate (called ES*) was found to precede ES1 in the pre-steady-state time period. The identity of the intermediates formed in aqueous solution with those previously observed in the cryostudy confirms that the mechanism is not changed either by the presence of an organic cosolvent or by subzero temperatures. Further characterization of ES1 and the steady-state intermediate ES2 at subzero temperatures, where their lifetime may be extended for up to several hours, has involved circular and magnetic circular dichroic studies. The magnetic circular dichroic spectra identify changes in the coordination sphere of the active-site metal during catalysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

Intramolecular distance measurements in alpha-lactalbumin

The distance between the calcium site (site I) and the zinc site (site II) in alpha-lactalbumin was estimated from Forster energy-transfer measurements between donor Eu(III) [or Tb(III)] at site I and acceptor Co(II) at site II to be 11.5 +/- 1.5 A. Intersite distances were also measured between the bis-ANS [4,4'-bis[1-(phenylamino)-8-naphthalenesulfonate]] binding locus and cobalt at site II (13.6 +/- 1.0 A), between bis-ANS and a fluorescein moiety covalently bound to Met-90 (33.5 +/- 3.0 A), and between Met-90 (fluorescein) and cobalt at site II (16.7 +/- 1.0 A). The apparent Kd for cobalt binding to site II agreed well with the value measured previously by intrinsic fluorescence [Murakami, K., & Berliner, L. J. (1983) Biochemistry 22, 3370-3374]. A Zn(II) titration of Eu(III)-alpha-lactalbumin reconfirmed that both sites I and II can be occupied simultaneously [Musci, G., & Berliner, L. J. (1985) Biochemistry 24, 3852-3856], since the lanthanide fluorescence was unaffected.     

Hydrolysis of peptides by carboxypeptidase A: equilibrium trapping of the ES2 intermediate

The cobalt absorption and electron paramagnetic resonance (EPR) spectra of cobalt carboxypeptidase undergo unique variations on formation of catalytic peptide and ester intermediates as previously recorded in cryoenzymologic experiments employing rapid-scanning spectroscopy and cryotrapping [Geoghegan, K. F., Galdes, A., Martinelli, R. A., Holmquist, B., Auld, D.S., & Vallee, B. L. (1983) Biochemistry 22, 2255-2262]. We here describe a means of stabilizing these intermediates, which we have termed "equilibrium trapping". It allows peptide intermediates to be observed for longer periods (much greater than 1 min) at ambient as well as subzero temperatures. The reaction intermediate with the rapidly turned over peptide substrate Dns-Ala-Ala-Phe is trapped when the cobalt enzyme (greater than 10 microM) has catalyzed the attainment of chemical equilibrium between high concentrations of the hydrolysis products Dns-Ala-Ala, 10 mM, and L-phenylalanine, 50 mM, and the product of their coupling Dns-Ala-Ala-Phe. Under these conditions, Dns-Ala-Ala-Phe is present in the equilibrated substrate-product reaction mixture at a level that exceeds the one predicted on the basis of K'eq for hydrolysis of this substrate and is close to the enzyme concentration. Other pairs of peptide hydrolysis products yield similar results. Visible absorption and EPR spectra of the cobalt enzyme show that the synthesized peptide binds to the active site in the mode previously recognized as the ES2 catalytic intermediate in peptide hydrolysis. Equilibrium trapping of the ES2 intermediate allows analysis of its physicochemical properties by methods that could not be employed readily under cryoenzymological conditions, e.g., circular dichroic and magnetic circular dichroic spectra.(ABSTRACT TRUNCATED AT 250 WORDS)     

Reconstitution of Escherichia coli ribosomes containing puromycin-modified S14: functional effects of the photoaffinity labeling of a protein essential for tRNA binding

In previous work we have shown that puromycin photoaffinity labels two proteins, L23 and S14, from separate sites of high affinity on Escherichia coli ribosomes [Jaynes, E. N., Jr., Grant, P. G., Giangrande, G., Wieder, R., & Cooperman, B. S. (1978) Biochemistry 17, 561-569; Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274], that puromycin-modified S14 is separable from native S14 by reverse-phase high-performance liquid chromatography (RP-HPLC), and that ribosomal proteins prepared by RP-HPLC can be reconstituted into active 30S subunits [Kerlavage, A. R., Weitzmann, C. J., & Cooperman, B. S. (1984) J. Chromatogr. 317, 201-212]. In this work we definitively identify puromycin-modified S14 by tryptic fingerprinting, an analysis that also provides evidence that the single tryptophan-containing peptide in S14 is the site of puromycin photoincorporation. We show that reconstituted 30S subunits, in which all of the S14 present is stoichiometrically modified with puromycin and all other ribosomal components are present in unmodified form, lack Phe-tRNAPhe binding activity and further that 70S ribosomes containing such reconstituted 30S subunits have substantially diminished binding activity to both the A and P sites, as differentiated through use of tetracycline. Suitable control experiments strongly indicate that this loss of activity is a direct consequence of puromycin photoincorporation.     

Effect of tubulin binding and self-association on the near-ultraviolet circular dichroic spectra of colchicine and analogues

Near-UV circular dichroic (CD) spectra of three colchicine analogues that differ at the C-10 position have been obtained in the presence and absence of tubulin. All three colchicine analogues show dramatic alterations in the low-energy near-UV CD band upon tubulin binding that cannot be mimicked by solvent, but in no event does the rotational strength of the CD band decrease to nearly zero as in the case of colchicine [Detrich, H. W., III, Williams, R. C., Jr., Macdonald, T. L., & Puett, D. (1981) Biochemistry 20, 5999-6005]. The effect of self-association of colchicine and one of the C-10 analogues, thiocolchicine, on the near-UV CD band was also investigated. A qualitative similarity was seen between the near-UV CD spectra of colchicine and thiocolchicine dimers and the spectra of these molecules bound to tubulin. These observations support the previous suggestion that ligands bound to the colchicine site on tubulin may be interacting with an aromatic amino acid in the colchicine binding site [Hastie, S. B., & Rava, R. P. (1989) J. Am. Chem. Soc. 110, 6993-7001].     

A differential scanning calorimetric study of the thermal unfolding of mutant forms of phage T4 lysozyme.

In continuation of our earlier work on the effects of amino acid replacements on the thermodynamics of the thermal unfolding of T4 lysozyme [Kitamura, S., & Sturtevant, J. M. (1989) Biochemistry 28, 3788-3792; Connelly, P., Ghosaini, L., Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1991) Biochemistry 30, 1887-1891; Hu, C.-Q., Kitamura, S., Tanaka, A., & Sturtevant, J. M. (1992) Biochemistry 31, 1643-1647], we report here a study by differential scanning calorimetry of the effects of five replacements at Ile3. Four of these replacements, those with Glu, Phe, Pro, and Thr, caused apparent destabilizations, while the replacement by Leu led to a small apparent stabilization. The largest observed destabilization (Ile3Pro) amounted to -3.0 kcal mol-1 in free energy at pH 2.00 and 38.8 degrees C (the denaturational temperature of the wild-type protein at this pH), and the largest stabilization amounted to +1.2 kcal mol-1 at pH 3.00 and 53.6 degrees C.     

Enzymatically inactive, exchange-inert Co(III)-carboxypeptidase A: role of inner sphere coordination in peptide and ester catalysis.

Catalytically inactive, exchange-inert Co(III)-carboxypeptidase A has been prepared by reaction of Co(II)-carboxypeptidase A with the active-site-directed oxidizing agent m-chloroperbenzoic acid. Co(III)-carboxypeptidase A, isolated by affinity gel filtration chromatography, has the same amino acid composition and molecular weight as the starting material and contains 0.95 g-atom/mol of cobalt and 0.01 g-atom/mol of zinc. Its electron paramagnetic resonance, circular dichroic, magnetic circular dichroic, and visible absorption spectra are consistent with those of octahedral Co(III) model complexes. Co(III)-caboxypeptidase A is essentially devoid of catalytic activity toward both peptide and ester substrates of the native enzyme, and stopped-flow fluorescence studies with dansylated substrates show that it binds peptides, but not esters. Furthermore, the protein does not react with either type of substrate to yield a single turnover. The implications of these findings to the mechanism of action of carboxypeptidase A are discussed in the light of the "metal-carbonyl" and "metal-hydroxide" hypotheses. Since Co(III)-carboxypeptidase A does not bind esters, inner-sphere coordination to the metal appears to be necessary for ester binding. All attempts to prepare Co(III)-carboxypeptidase A by treatment of Co(II)-carboxypeptidase A with hydrogen peroxide according to previously published procedures (Kang, E.P., Storm, C.B., & Carson, F.W. (1975) J. Am. Chem. Soc. 97, 6723) have been unsuccessful, and the present results do not confirm earlier reports that Co(III)-carboxypeptidase A exhibits esterase activity or that its activity is dependent on the method of preparation of the precursor Co(II)-carboxypeptidase A (Jones, M.M., Hunt, J.B., Storm, C.B., Evans, P.S., Carson, F.W. & Pauli, W.J. (1977) Biochem. Biophys. Res. Commun. 75, 253). These findings call for a reexamination of mechanistic conclusions based on the assumption that Co(III)-carboxypeptidase A is an active esterase.     

Comparison of the data, analysis, and results of X-ray absorption studies of cytochrome c oxidase

Differences in the methods of analysis of X-ray absorption data used by Powers et al. [Powers, L., Blumberg, W. E., Chance, B., Barlow, C., Leigh, J., Jr., Smith, J., Yonetani, T., Vik, S., & Peisach, J. (1979) Biochim. Biophys. Acta 547, 520-538; Powers, L., Chance, B., Ching, Y., & Angiolillo, P. (1981) Biophys. J. 34, 465-498] and Scott et al. [Scott, R., Schwartz, J., & Cramer S. (1986) Biochemistry 25, 5546-5555] are clarified. In addition, we compare the X-ray absorption data and results for resting cytochrome c oxidase reported by both groups using the same analysis method and conclude apart from any assumptions that the data are not identical.     

Secondary structure of human plasma fibronectin: conformational change induced by calf alveolar heparan sulfates

The quantitative analysis of circular dichroic spectra of native human plasma fibronectin according to the method of Provencher and Gl?ckner [Provencher, S. W., & Gl?ckner, J. (1981) Biochemistry 20, 33-37] indicated the presence of beta-sheet (79%), beta-turn (21%), but no alpha-helix or random coil in the secondary structure. The calf alveolar heparan sulfates induced a change in the conformation of fibronectin: the magnitude of the change depended on the molecular properties of the particular heparan sulfate preparations.     

Structural influence of hydrophobic core residues on metal binding and specificity in carbonic anhydrase II

Aromatic residues in the hydrophobic core of human carbonic anhydrase II (CAII) influence metal ion binding in the active site. Residues F93, F95, and W97 are contained in a beta-strand that also contains two zinc ligands, H94 and H96. The aromatic amino acids contribute to the high zinc affinity and slow zinc dissociation rate constant of CAII [Hunt, J. A., and Fierke, C. A. (1997) J. Biol. Chem. 272, 20364-20372]. Substitution of these aromatic amino acids with smaller side chains enhances Cu(2+) affinity while decreasing Co(2+) and Zn(2+) affinity [Hunt, J. A., Mahiuddin, A., & Fierke, C. A. (1999) Biochemistry 38, 9054-9062]. Here, X-ray crystal structures of zinc-bound F93I/F95M/W97V and F93S/F95L/W97M CAIIs reveal the introduction of new cavities in the hydrophobic core, compensatory movements of surrounding side chains, and the incorporation of buried water molecules; nevertheless, the enzyme maintains tetrahedral zinc coordination geometry. However, a conformational change of direct metal ligand H94 as well as indirect (i.e., "second-shell") ligand Q92 accompanies metal release in both F93I/F95M/W97V and F93S/F95L/W97M CAIIs, thereby eliminating preorientation of the histidine ligands with tetrahedral geometry in the apoenzyme. Only one cobalt-bound variant, F93I/F95M/W97V CAII, maintains tetrahedral metal coordination geometry; F93S/F95L/W97M CAII binds Co(2+) with trigonal bipyramidal coordination geometry due to the addition of azide anion to the metal coordination polyhedron. The copper-bound variants exhibit either square pyramidal or trigonal bipyramidal metal coordination geometry due to the addition of a second solvent molecule to the metal coordination polyhedron. The key finding of this work is that aromatic core residues serve as anchors that help to preorient direct and second-shell ligands to optimize zinc binding geometry and destabilize alternative geometries. These geometrical constraints are likely a main determinant of the enhanced zinc/copper specificity of CAII as compared to small molecule chelators.     

Multinuclear magnetic resonance studies of the 2Fe.2S* ferredoxin from Anabaena species strain PCC 7120. 2. Sequence-specific carbon-13 and nitrogen-15 resonance assignments of the oxidized form

Multinuclear two-dimensional NMR techniques were used to assign nearly all diamagnetic 13C and 15N resonances of the plant-type 2Fe.2S* ferredoxin from Anabaena sp. strain PCC 7120. Since a 13C spin system directed strategy had been used to identify the 1H spin systems [Oh, B.-H., Westler, W. M., & Markley, J. L. (1989) J. Am. Chem. Soc. 111, 3083-3085], the sequence-specific 1H assignments [Oh, B.-H., & Markley, J. L. (1990) Biochemistry (first paper of three in this issue)] also provided sequence-specific 13C assignments. Several resonances from 1H-13C groups were assigned independently of the 1H assignments by considering the distances between these nuclei and the paramagnetic 2Fe.2S* center. A 13C-15N correlation data set was used to assign additional carbonyl carbons and to analyze overlapping regions of the 13C-13C correlation spectrum. Sequence-specific assignments of backbone and side-chain nitrogens were based on 1H-15N and 13C-15N correlations obtained from various two-dimensional NMR experiments.     

Reconstitution of Escherichia coli 50S ribosomal subunits containing puromycin-modified L23: functional consequences

In previous work we have shown that both puromycin [Weitzmann, C. J., & Cooperman, B. S. (1985) Biochemistry 24, 2268-2274] and p-azidopuromycin [Nicholson, A. W., Hall, C. C., Strycharz, W. A., & Coooperman, B. S. (1982) Biochemistry 21, 3809-3817] site specifically photoaffinity label protein L23 to the highest extent of any Escherichia coli ribosomal protein. In this work we demonstrate that L23 that has been photoaffinity labeled within a 70S ribosome by puromycin (puromycin-L23) can be separated from unmodified L23 by reverse-phase high-performance liquid chromatography (RP-HPLC) and further that puromycin-L23 can reconstitute into 50S subunits when added in place of unmodified L23 to a reconstitution mixture containing the other 50S components in unmodified form. We have achieved a maximum incorporation of 0.5 puromycin-L23 per reconstituted 50S subunit. As compared with reconstituted 50S subunits either containing unmodified L23 or lacking L23, reconstituted 50S subunits containing 0.4-0.5 puromycin-L23 retain virtually all (albeit low) peptidyl transferase activity but only 50-60% of mRNA-dependent tRNA binding stimulation activity. We conclude that although L23 is not directly at the peptidyl transferase center, it is sufficiently close that puromycin-L23 can interfere with tRNA binding. This conclusion is consistent with a number of other experiments placing L23 close to the peptidyl transferase center but is difficult to reconcile with immunoelectron microscopy results placing L23 near the base of the 50S subunit on the side facing away from the 30S subunit [Hackl, W., & St?ffler-Meilicke, M. (1988) Eur. J. Biochem. 174, 431-435].     

NMR studies of the AMP-binding site and mechanism of adenylate kinase

NMR has previously been used to determine the conformation of enzyme-bound MgATP and to locate the MgATP-binding site on adenylate kinase [Fry, D. C., Kuby, S. A., & Mildvan, A. S. (1985) Biochemistry 24, 4680-4694]. To determine the conformation and location of the other substrate, AMP, distances have been measured from Cr3+AMPPCP, a linear competitive inhibitor with respect to MgATP, to six protons and to the phosphorus atom of AMP on adenylate kinase, with the paramagnetic probe-T1 method. Time-dependent nuclear Overhauser effects (NOEs) have been used to measure five interproton distances on enzyme-bound AMP. These distances were used to determine the conformation of bound AMP in addition to its position with respect to metal-ATP. Enzyme-bound AMP exhibits a high anti-glycosyl torsional angle (chi = 110 +/- 10 degrees), a 3'-endo,2'-exo ribose pucker (delta = 105 +/- 10 degrees), and gauche-trans orientations about the C4'-C5' bond (gamma = 180 +/- 10 degrees) and the C5'-O5' bond (beta = 170 +/- 20 degrees). The distance from Cr3+ to the phosphorus of AMP is 5.9 +/- 0.3 A, indicating a reaction coordinate distance of approximately 3 A, which is consistent with an associative SN2 mechanism for the phosphoryl transfer. Ten intermolecular NOEs, from protons of the enzyme to those of AMP, were detected, indicating the proximity of at least three hydrophobic amino acids to bound AMP. These constraints, together with the conformation of AMP and the intersubstrate distances, were used to position AMP into the X-ray structure of adenylate kinase. The AMP binding site is found to be near (less than or equal to 4 A from) Leu-116, Arg-171, Val-173, Val-182, and Leu-190; all of these residues have been found to be invariant in muscle-type rabbit, calf, human, porcine [Kuby, S. A., Palmieri, R. H., Frischat, A., Fischer, A. H., Wu, L. H., Maland, L., & Manship, M. (1984) Biochemistry 23, 2393-2399], and chicken adenylate kinase [Kishi, F., Maruyama, M., Tanizawa, Y., & Nakazawa, A. (1986) J. Biol. Chem. 261, 2942-2945].     

Nuclear Overhauser effect studies of the conformations of MgATP bound to the active and secondary sites of muscle pyruvate kinase

MgATP binds both at the active site (site 1) and at a secondary site (site 2) on each monomer of muscle pyruvate kinase as previously found by binding studies and by X-ray analysis. Interproton distances on MgATP bound at each site have been measured by the time-dependent nuclear Overhauser effect in the absence and presence of phosphoenolpyruvate (P-enolpyruvate), which blocks ATP binding at site 1. Interproton distances at site 2 are consistent with a single conformation of bound ATP with a high antiglycosidic torsional angle (chi = 68 +/- 10 degrees) and a C3'-endo ribose pucker (delta = 90 +/- 10 degrees). Interproton distances at site 1, determined in the absence of P-enolpyruvate by assuming the averaging of distances at both sites, cannot be fit by a single adenine-ribose conformation but require the contribution of at least three low-energy structures: 62 +/- 10% low anti (chi = 30 degrees), C3'-endo; 20 +/- 8% high anti (chi = 55 degrees), O1'-endo; and 18 +/- 8% syn (chi = 217 degrees), C2'-endo. Although a different set of ATP conformations might also have fit the interproton distances, the mixture of conformations used also fits previously determined distances from Mn2+ to the protons of ATP bound at site 1 [Sloan, D. L., & Mildvan, A. S. (1976) J. Biol. Chem. 251, 2412] and is similar to the adenine-ribose portion of free Co(NH3)4ATP, which consists of 35% low anti, 51% high anti, and 14% syn [Rosevear, P. R., Bramson, H. N., O'Brian, C., Kaiser, E. T., & Mildvan, A. S. (1983) Biochemistry 22, 3439].(ABSTRACT TRUNCATED AT 250 WORDS)     

Phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate: synthesis, purification, and partial characterization

The 1-phosphorothioate analogues of 5-phosphoribosyl 1-diphosphate (P-Rib-PP) have been prepared enzymatically, in reactions catalyzed by P-Rib-PP synthetase from Salmonella typhimurium. 5-Phosphoribosyl 1-O-(2-thiodiphosphate) (P-Rib-PP beta S) was synthesized from ribose 5-phosphate (Rib-5-P) and the Mg2+ complex of adenosine 5'-O-(3-thiotriphosphate). The SP and RP diastereomers of 5-phosphoribosyl 1-O-(1-thiodiphosphate) (P-Rib-PP alpha S) were synthesized from Rib-5-P and the Mg2+ complex of adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) (SP diastereomer, delta-configuration) and the Cd2+ complex of ATP beta S (RP diastereomer, delta-configuration), respectively. The strategy for the synthesis and stereochemical assignment of the P-Rib-PP alpha S diastereomers was based on the specificity of P-Rib-PP synthetase for the (delta)-beta, gamma-bidentate metal-nucleotide substrate and the stereochemical course of the synthetase reaction, leading to inversion of configuration at the P beta atom of the nucleotide [Li, T. M., Mildvan, A. S., & Switzer, R. L. (1978) J. Biol. Chem. 253, 3918-3923], and the known configurations of the Mg2+ and Cd2+ beta, gamma-bidentate complexes of the ATP beta S diastereomers [Jaffe, E. K., & Cohn, M. (1979) J. Biol. Chem. 254, 10839-10845]. The P-Rib-PP analogues were purified by gradient elution from DEAE-Sephadex and characterized by chemical analysis and 31P nuclear magnetic resonance [Smithers, G. W., & O'Sullivan, W. J. (1984) Biochemistry (following paper in this issue)]. A preliminary account of their interaction with human brain hypoxanthine phosphoribosyltransferase and yeast orotate phosphoribosyltransferase (OPRTase) is described.(ABSTRACT TRUNCATED AT 250 WORDS)     

Solution studies of staphylococcal nuclease H124L. 2. 1H, 13C, and 15N chemical shift assignments for the unligated enzyme and analysis of chemical shift changes that accompany formation of the nuclease-thymidine 3',5'-bisphosphate-calcium ternary complex.

Accurate 1H, 15N, and 13C chemical shift assignments were determined for staphylococcal nuclease H124L (in the absence of inhibitor or activator ion). Backbone 1H and 15N assignments, obtained by analysis of three-dimensional 1H-15N HMQC-NOESY data [Wang, J., Mooberry, E.S., Walkenhorst, W.F., & Markley, J. L. (1992) Biochemistry (preceding paper in this issue)], were refined and extended by a combination of homo- and heteronuclear two-dimensional NMR experiments. Staphylococcal nuclease H124L samples used in the homonuclear 1H NMR studies were at natural isotopic abundance or labeled randomly with 2H (to an isotope level of 50%); nuclease H124L samples used for heteronuclear NMR experiments were labeled uniformly with 15N (to an isotope level greater than 95%) or uniformly with 13C (to an isotope level of 26%). Additional nuclease H124L samples were labeled selectively by incorporating single 15N- or 13C-labeled amino acids. The chemical shifts of uncomplexed enzyme were then compared with those determined previously for the nuclease H124L.pdTp.Ca2+ ternary complex [Wang, J., LeMaster, D. M., & Markley, J.L. (1990) Biochemistry 29, 88-101; Wang, J., Hinck, A.P., Loh, S. N., & Markley, J.L. (1990) Biochemistry 29, 102-113; Wang, J., Hinck, A.P., Loh, S.N., & Markley, J.L. (1990) Biochemistry 29, 4242-4253]. The results reveal that the binding of pdTp and Ca2+ induces large shifts in the resonances of several amino acid segments. These chemical shift changes are interpreted in terms of changes in backbone torsion angles that accompany the binding of pdTp and Ca2+; changes at the binding site appear to be transmitted to other regions of the molecule through networks of hydrogen bonds.     

Isolation and characterization of 101-beta-lysozyme that possesses the beta-aspartyl sequence at aspartic acid-101

In the reaction of the intramolecular cross-linking between Lys-13 (epsilon-NH3+) and Leu-129 (alpha-COO-) in lysozyme using imidazole and 1-ethyl-3-[3-(dimethylamino)propyl]carbodiimide hydrochloride [Yamada, H., Kuroki, R., Hirata, M., & Imoto, T. (1983) Biochemistry 22, 4551-4556], it was found that two-thirds of the protein (both the recovered and cross-linked lysozymes) showed a lower affinity than the rest against chitin-coated Celite, an affinity adsorbent for lysozyme. The protein with the reduced affinity was separated on chitin-coated Celite affinity chromatography and found to be slightly different from native lysozyme in the elution position of the tryptic peptide of Ile-98-Arg-112 on reversed-phase high-performance liquid chromatography. In contrast with native lysozyme, the limited hydrolysis of this abnormal tryptic peptide of Ile-98-Arg-112 in 6 N HCl at 110 degrees C gave a considerable amount of beta-aspartylglycine. Therefore, it was concluded that two-thirds of the protein obtained from this reaction possessed the beta-aspartylglycyl sequence at Asp-101-Gly-102. As a result, we obtained four lysozymes from this reaction, the derivative with the beta-aspartyl sequence at Asp-101 (101-beta-lysozyme), the cross-linked derivative between Lys-13 and Leu-129 (CL-lysozyme), the CL-lysozyme derivative with the beta-aspartyl sequence at Asp-101 (101-beta-CL-lysozyme), and native lysozyme. In the ethyl esterification of Asp-52 in lysozyme with triethyloxonium fluoroborate [Parsons, S. M., Jao, L., Dahlquist, F. W., Borders, C. L., Jr., Groff, T., Racs, J., & Raftery, M. A. (1969) Biochemistry 8, 700-712; Parsons, S. M., & Raftery, M. A. (1969) Biochemistry 8, 4199-4205], the same bond rearrangement was detected in the same ratio.(ABSTRACT TRUNCATED AT 250 WORDS)     

Linkage of organic phosphates to oxygen binding in human hemoglobin at high concentrations

We have performed high-precision oxygen binding studies on human hemoglobin tetramers in the presence of a series of limited, subsaturating amounts of the effector compounds 2,3-diphosphoglycerate (DPG) and inositol hexaphosphate (IHP). The use of thin-layer optical methods enabled the use of high hemoglobin concentrations, preventing complications arising from the dissociation of the tetramer into dimers. Model-independent, simultaneous analysis of all data for each effector demonstrated that the intrinsic oxygen binding characteristics of the molecule are in agreement with those determined in earlier high-precision studies [e.g., Gill, S. J., Di Cera, E., Doyle, M. L., Bishop, G. A., & Robert, C. H. (1987) Biochemistry 26, 3995-4002] and that the affinity of the tetramer for the tightly binding effector IHP changes most markedly between the second and fourth oxygen binding steps, perhaps indicating a large conformational change. The data were then analyzed by using the truncated allosteric model [Di Cera, E., Robert, C. H., & Gill, S. J. (1987) Biochemistry 26, 4003-4008], which is based on the hypothesis that a quaternary conformational change occurs in the hemoglobin tetramer before the third and fourth oxygen molecules bind.