Kinetic mechanism of a partial folding reaction. 1. Properties Of the reaction and effects of denaturants

The bimolecular association rate constant (kon) and dissociation rate constant (koff) of the complex between fluorescein-labeled S-peptide analogues and folded S-protein are reported. This is the first kinetic study of a protein folding reaction in which most of the starting material is already folded and only a small part (one additional helix) becomes ordered; it provides a folding landscape with a small conformational entropy barrier, and one in which kinetic traps are unlikely. Refolding and unfolding are measured under identical strongly native conditions, and the reaction is found to be two-state at low reactant concentrations. The dissociation constant (Kd) of the complex and the properties of the transition state may be calculated from the rate constants without extrapolation. The folded complex is formed fast (kon = 1.8 x 10(7) M-1 s-1) and is very stable (Kd = 6 pM) at 10 degrees C, 10 mM MOPS, pH 6.7. Charge interactions stabilize the complex by 1.4 kcal mol-1. The charge effect enters in the refolding reaction: increasing the salt concentration reduces kon dramatically and has little effect on koff. Urea and GdmCl destabilize the complex by decreasing kon and increasing koff. The slopes (m-values) of plots of ln Kd vs [cosolvent] are 0.75 +/- 0.04 and 2.8 +/- 0.3 kcal mol-1 M-1 for urea and GdmCl, respectively. The ratio mon/(mon + moff) is 0.54 +/- 0.04 for urea and 0.57 +/- 0.1 for GdmCl, where mon is the m-value for kon and moff is the m-value for koff, indicating that more than half of the sites for interaction with either cosolvent are buried in the ensemble of structures present at the transition state.     

Structure, interaction and electron transfer between cytochrome b5, its E44A and/or E56A mutants and cytochrome c

Site-directed mutagenesis has been used to produce variants of a tryptic fragment of bovine liver cytochrome b5 in which Glu44 and Glu56 are mutated to alanine. The reduction potentials measured by spectroelectrochemical titration (in the presence of 1 mM (Ru(NH3)6)3+, pH 7.0 and I=0.1 M) are 4.5, 6.0, 6.0 and 7.5 mV versus the standard hydrogen electrode (SHE) for the wild-type and E44A, E56A and E44/56A mutants of cytochrome b5, respectively. A comparative two-dimensional NMR study of cytochrome b5 and its E44/56A mutant in water solution has been achieved. Resonance assignments of side-chains have been completed successfully. The NMR results suggest that the secondary structures and global folding of the E44/56A mutant remain unchanged, but the mutation of both Glu44 and Glu56 to hydrophobic alanine may lead to the two helices containing mutated residues contracting towards the heme center. The inner mobility of the Gly42 approximately Glu44 segment in cytochrome b5 may be responsible for the difference of the binding mode between Glu44 and Glu56 with cytochrome c. The binding between cytochrome c and cytochrome b5 was studied by optical difference spectra of cytochrome c and variants of cytochrome b5. The association constants (KA) for the wild-type, E44A, E56A, and E44/56A mutants of cytochrome b5 with cytochrome c, are 4.70(+/-0. 10)x10(6) M-1, 1.88(+/-0.03)x10(6) M-1, 2.70(+/-0.13)x10(6) M-1, and 1.14(+/-0.05)x10(6) M-1, respectively. This is indicative that both Glu44 and Glu56 are involved in the complex formation between cytochrome b5 and cytochrome c. The reduction of horse heart ferricytochrome c by recombinant ferrocytochrome b5 and its mutants has been studied. The rate constant of the electron transfer reaction between ferricytochrome c and wild-type ferrocytochrome b5 (1.074(+/-0.49)x10(7) M-1 s-1) is higher than those of the mutant protein E44A (8.98(+/-0.20)x10(6) M-1 s-1), E56A (8.76(+/-0. 39)x10(6) M-1 s-1), and E44/56A (8.02(+/-0.38)x10(6) M-1 s-1) at 15 degreesC, pH 7.0, I=0.35 M. The rate constants are strongly dependent on ionic strength and temperature. These studies, by means of a series of techniques, provide conclusive results that the interaction between cytochrome b5 and cytochrome c is electrostatically guided, and, more importantly, that both Glu44 and Glu56 participate in the electron transfer reaction.     

Peptide-induced conformational changes in the molecular chaperone DnaK

DnaK, the 70 kDa molecular chaperone of Escherichia coli, adopts a high-affinity state in the presence of ADP that tightly binds its target peptide, whereas replacement of ADP by ATP induces a structural switch to a low-affinity chaperone state that weakly binds its target. An approximately 15% decrease in tryptophan fluorescence of DnaK occurs in concert with this switch from the high- to low-affinity state. The reversibility of this structural transition in DnaK was investigated using rapid mixing and equilibrium fluorescence methods. The Cro peptide (MQERITLKDYAM) was used to mimic an unfolded substrate. When the Cro peptide is rapidly mixed with preformed low-affinity DnaK complexes (DnaK-ATP), a rapid increase (kobs = 3-30 s-1) in the tryptophan fluorescence of DnaK occurs. We suggest that the Cro peptide induces the transition of the low-affinity state of DnaK back to the high-affinity state, without ATP hydrolysis. The combined results in this report are consistent with the minimal mechanism ATP + EP if ATP-EP if ATP-E + P, where ATP binding (K1) induces a conformational change and concerted peptide release (koff), and peptide binding (kon) to the low-affinity state (ATP-E) induces the transition back to ATP-EP, a high-affinity state. At 25 degreesC, in the presence of the Cro peptide, values for K1, koff, and kon are 22 microM, 3.3 s-1, and 2. 4 x 10(4) M-1 s-1, respectively. Evidence for an equilibrium between closed and open forms of DnaK in the absence of ATP and peptide is also presented.     

Transient kinetics of formation and reaction of the uridylyl-enzyme form of galactose-1-P uridylyltransferase and its Q168R-variant: insight into the molecular basis of galactosemia

Galactose-1-phosphate uridylyltransferase catalyzes the reaction of UDP-glucose with galactose 1-phosphate (Gal-1-P) to form UDP-galactose and glucose 1-phosphate (Glc-1-P) through a double displacement mechanism, with the intermediate formation of a covalent uridylyl-enzyme (UMP enzyme). Gln 168 in E. coli uridylyltransferase engages in hydrogen bonding with the phosphoryl oxygens of the UMP moiety, which is bonded to His 166 in the intermediate [Wedekind, J. E., Frey, P. A., and Rayment, I. (1996) Biochemistry 35, 11560-11569]. In humans, the point variant Q188R accounts for 60% of galactosemia cases. The corresponding E. coli variant Q168R has been overexpressed and purified. In preparation for kinetic correlation of Q168R and wild-type uridylyltransferases, we tested the kinetic competence of the wild-type UMP-enzyme. At 4 degreesC, the first-order rate constant for uridylylation by UDP-glucose is 281 +/- 18 s-1, and for deuridylylation it is 226 +/- 10 s-1 with Glc-1-P and 166 +/- 10 s-1 with Gal-1-P. Inasmuch as the overall turnover number at 4 degreesC is 62 s-1, the covalent intermediate is kinetically competent. The variant Q168R is uridylylated by UDP-glucose to the extent of about 65% of the potential active sites. Uridylylation reactions of Q168R with UDP-glucose proceed with maximum first-order rate constants of 2.2 x 10(-)4 s-1 and 4.2 x 10(-)4 s-1 at 4 and 27 degreesC, respectively. In experiments with uridylyl-Q168R and glucose-1-P, the mutant enzyme undergoes deuridylylation with maximum first-order rate constants of 4.8 x 10(-)4 s-1 and 1.68 x 10(-)3 s-1 at 4 and 27 degreesC, respectively. The value of Km for uridylylation of Q168R is slightly higher than for the wild-type enzyme, and for deuridylylation it is similar to the wild-type value. The wild-type enzyme undergoes uridylylation and deuridylyation about 10(6) times faster than Q168R. The wild-type activity in the overall reaction is 1.8 x 10(6) times that of Q168R. The wild-type enzyme contains 1.9 mol of Zn+Fe per mole of subunits, whereas the Q168R-variant contains 1.36 mol of Zn+Fe per mole of subunits. The mutation stabilizes the uridylyl-enzyme by 1.2 kcal mol-1 in comparison to the wild-type enzyme. These results show that the low activity of Q168R is not due to overstabilization of the intermediate or to the absence of structural metal ions. Instead, the main defect is very slow uridylylation and deuridylation.     

Kinetic study of the reaction of glutathione peroxidase with peroxynitrite

Glutathione peroxidases and their mimics, e.g., ebselen or diaryl tellurides, efficiently reduce peroxynitrite/peroxynitrous acid (ONOO-/ONOOH) to nitrite and protect against oxidation and nitration reactions. Here, we report the second-order rate constant for the reaction of the reduced form of glutathione peroxidase (GPx) with peroxynitrite as (8.0 +/- 0.8) x 10(6) M-1 s-1 (per GPx tetramer) at pH 7.4 and 25 degreesC. The rate constant for oxidized GPx is about 10 times lower, (0.7 +/- 0.2) x 10(6) M-1 s-1. On a selenium basis, the rate constant for reduced GPx is similar to that obtained previously for ebselen. The data support the conclusion that GPx can exhibit a biological function by acting as a peroxynitrite reductase.     

Rat liver transcript profiling in normal and disease states using a kinetic polymerase chain reaction assay

The retention rate of the spin label 3-isothiocyanto methyl-2,2,5,5-tetramethyl-1-pyrrolidinyl oxyl spin label (proxyl) attached to the porcine N-acetyl-NPY peptide and the porcine N-acetyl-D-Trp32-NPY peptide at Lys4 was investigated using SK-N-MC neuroblastoma cell membranes containing the Y1 receptor. The release rate of the spin labeled peptides was monitored by electron spin resonance and the KD was determined by a direct radiolabeled NPY displacement binding assay. The analyses show that for the porcine [Ac-Tyr1N epsilon 4-proxyl]-NPY, the KD was 8 x 10(-10) M and koff was 2.7 x 10(-4) sec-1 yielding a value for kon of 3.3 x 10(5) sec-1 M-1. The [Ac-Tyr1, N epsilon 4-proxyl,-D-Trp32]-NPY antagonist ligand had a value of KD equal to 1.35 x 10(-7) M and koff was 1.7 x 10(-4) sec-1 leading to a value for kon of 1.2 x 10(3) sec-1 M-1. The difference in the kon rates of two orders of magnitude is interpreted as demonstrating the N-acetyl-N epsilon 4 proxyl-D-Trp32-NPY ligand binding transition state to be of higher energy then for the unmodified NPY amino acid sequence.     

Affinity and kinetic analysis of P-selectin binding to P-selectin glycoprotein ligand-1

Leukocytes use the cell-surface mucin P-selectin glycoprotein ligand-1 (PSGL-1) to tether to and roll on P-selectin on activated endothelial cells and platelets. By using surface plasmon resonance, we measured the affinity and kinetics of binding of soluble monomeric human P-selectin to immobilized PSGL-1 from human neutrophils. Binding was specific, as documented by its Ca2+-dependence, its inhibition by specific monoclonal antibodies to P-selectin and PSGL-1, and its abrogation by treating PSGL-1 with sialidase. Similar binding was observed for soluble P-selectin that contained the lectin and epidermal growth factor domains plus all nine consensus repeats, and for a soluble construct that contained only the lectin and epidermal growth factor domains. Soluble P-selectin bound saturably to a single class of sites on PSGL-1 with a dissociation constant (Kd) of 320 +/- 20 nM. The measured koff was 1.4 +/- 0.1 s-1, and the calculated kon was 4.4 x 10(6) M-1 s-1. We conclude that monomeric P-selectin binds to PSGL-1 with fast association and dissociation rates and relatively high affinity. These features may be important for efficient tethering and rolling of leukocytes at physiologic densities of PSGL-1 and P-selectin.     

Subconductance block of single mechanosensitive ion channels in skeletal muscle fibers by aminoglycoside antibiotics

The activity of single mechanosensitive channels was recorded from cell-attached patches on acutely isolated skeletal muscle fibers from the mouse. The experiments were designed to investigate the mechanism of channel block produced by externally applied aminoglycoside antibiotics. Neomycin and other aminoglycosides reduced the amplitude of the single-channel current at negative membrane potentials. The block was concentration-dependent, with a half-maximal concentration of approximately 200 microM. At high drug concentrations, however, block was incomplete with roughly one third of the current remaining unblocked. Neomycin also caused the channel to fluctuate between the open state and a subconductance level that was also roughly one third the amplitude of the fully open level. An analysis of the kinetics of the subconductance fluctuations was consistent with a bimolecular reaction between an aminoglycoside molecule and the open channel (kon = approximately 1 x 10(6) M-1s-1 and koff = approximately 400 s-1 at -60 mV). Increasing the external pH reduced both the rapid block of the open channel and the frequency of the subconductance fluctuations, as if both blocking actions were produced by a single active drug species with a pKa = approximately 7.5. The results are interpreted in terms of a mechanism in which an aminoglycoside molecule partially occludes ion flow through the channel pore.     

Purification and preliminary characterization of a serine hydrolase involved in the microbial degradation of polychlorinated biphenyls

2-Hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA) hydrolase (BphD), an enzyme of the biphenyl biodegradation pathway encoded by the bphD gene of Burkholderia cepacia LB400, was hyperexpressed and purified to apparent homogeneity. SDS-polyacrylamide gel electrophoresis confirmed that BphD has a subunit molecular mass of 32 kDa, while gel filtration demonstrated that it is a homotetramer of molecular weight 122,000. The enzyme hydrolyzed 6-phenyl-HODA with a kcat of 5.0 (+/- 0.07) s-1 and a kcat/Km of 2.0 (+/- 0.08) x 10(7) M-1 s-1 (100 mM phosphate, pH 7.5, 25 degreesC). The specificity of BphD for other 2-hydroxy-6-oxohexa-2,4-dienoates (HODAs) decreased markedly with the size of the C6 substituent; 6-methyl-HODA, the meta cleavage product of 3-methylcatechol, was hydrolyzed approximately 2300 times less specifically than 6-phenyl-HODA. By comparison, the homologous hydrolase from the toluene degradation pathway, TodF, showed highest specificity for 6-methyl- and 6-ethyl-HODA (kcat/Km of 2.0 (+/- 0.05) x 10(6) M-1 s-1 and 9.0 (+/- 0.5) x 10(6) M-1 s-1, respectively). TodF showed no detectable activity toward 6-phenyl-HODA and 6-tert-butyl-HODA. Neither BphD nor TodF hydrolyzed 5-methyl-HODA efficiently. The kcat of BphD determined by monitoring product formation was about half that determined by monitoring substrate disappearance, suggesting that some uncoupling of substrate utilization and product formation occurs during the enzyme catalyzed reaction. Crystals of BphD were obtained using ammonium sulfate combined with polyethylene glycol 400 as the precipitant. Diffraction was observed to a resolution of at least 1.9 A, and the evaluation of self-rotation functions confirmed 222 (D2) molecular symmetry.     

Identification of tissue-type plasminogen activator-specific plasminogen activator inhibitor-1 mutants. Evidence that second sites of interaction contribute to target specificity

Plasminogen activator inhibitor-1 (PAI-1) is the primary inhibitor of the plasminogen activators (PAs), tissue-type plasminogen activator (tPA), and urokinase-type plasminogen activator (uPA). A library of PAI-1 mutants containing substitutions at the P1 and P1' positions was screened for functional activity against tPA and thrombin. Several PAI-1 variants that were inactive against uPA in a previous study (Sherman, P. M., Lawrence, D. A., Yang, A. Y., Vandenberg, E. T., Paielli, D., Olson, S. T., Shore, J. D., and Ginsburg, D. (1992) J. Biol. Chem. 267, 7588-7595) had significant inhibitory activity toward tPA. This set of tPA-specific PAI-1 mutants contained a wide range of amino acid substitutions at P1 including Asn, Gln, His, Ser, Thr, Leu, Met, and all the aromatic amino acids. This group of mutants also demonstrated a spectrum of substitutions at P1'. Kinetic analyses of selected variants identified P1Tyr and P1His as the most efficient tPA-specific inhibitors, with second-order rate constants (ki) of 4.0 x 10(5) M-1s-1 and 3.6 x 10(5) M-1s-1, respectively. Additional PA-specific PAI-1 variants containing substitutions at P3 through P1' were constructed. P3Tyr-P2Ser-P1Lys-P1'Trp and P3Tyr-P2Ser-P1Tyr-P1'Met had ki values of 1.7 x 10(6) M-1s-1 and 2.5 x 10(6) M-1s-1 against tPA, respectively, but both were inactive against uPA. In contrast, P2Arg-P1Lys-P1'Ala inhibited uPA 74-fold more rapidly than tPA. The mutant PAI-1 library was also screened for inhibitory activity toward thrombin in the presence and absence of the cofactor heparin. While wild-type PAI-1 and several P1Arg variants inhibited thrombin in the absence of heparin, a number of variants were thrombin inhibitors only in the presence of heparin. These results demonstrate the importance of the reactive center residues in determining PAI-1 target specificity and suggest that second sites of interaction between inhibitors and proteases can also contribute to target specificity. Finally, the PA-specific mutants described here should provide novel reagents for dissecting the physiological role of PAI-1 both in vitro and in vivo.     

Recognition between disordered states: kinetics of the self-assembly of thioredoxin fragments

The disordered N- (1-73) and C- (74-108) fragments of oxidized Escherichia colithioredoxin (Trx) reconstitute the native structure upon association [Tasayco, M. L., & Chao, K. (1995) Proteins: Struct., Funct., Genet. 22, 41-44]. Kinetic measurements of the formation of the complex (1-73/74-108) at 20 degrees C under apparent pseudo-first-order conditions using stopped-flow far-UV CD and fluorescence spectroscopies indicate association coupled to folding, an apparent rate constant of association [kon = (1330 +/- 54) M-1 s-1], and two apparent unimolecular rate constants [k1 = (0. 037 +/- 0.007) s-1 and k2 = (0.0020 +/- 0.0005) s-1]. The refolding kinetics of the GuHCl denatured Trx shows the same two slowest rate constants. An excess of N- over C-fragment decreases the kon, and the slowest phase disappears when a P76A variant is used. Stopped-flow fluorescence measurements at 20 degrees C indicate a GuHCl-dependent biphasic dissociation/unfolding process of the complex, where the slowest phase corresponds to 90% of the total. Their rate constants, extrapolated to zero denaturant, k-1 = (9 +/- 3) x 10(-5) s-1 and k-2 = (3.4 +/- 1.2) x 10(-5) s-1, show m# values of (4.0 +/- 0.4) kcal mol-1 M-1 and (3.5 +/- 0.1) kcal mol-1 M-1, respectively. Our results indicate that: (i) a compact intermediate with trans P76 and defined tertiary structure seems to participate in both the folding and unfolding processes; (ii) not all the N-fragment is competent to associate with the C-fragment; (iii) conversion to an association competent form occurs apparently on the time scale of P76 isomerization; and (iv) the P76A variation does not alter the association competency of the C-fragment, but it permits its association with "noncompetent" forms of the N-fragment.     

Imidazole is a sensitive probe of steric hindrance in the distal pockets of oxygen-binding heme proteins

The FixL heme-based sensor, despite its low affinity for oxygen, is much more reactive than myoglobin toward the large polar ligand imidazole. To determine which features of a myoglobin heme pocket favor binding of imidazole, we have measured binding of this ligand to the FixL heme domain, elephant myoglobin, wild-type sperm whale myoglobin, and sperm whale myoglobins having alanine, valine, threonine, glutamine, leucine, phenylalanine, or tryptophan substitutions of the distal (E7) histidine residue. Except for histidine, the association rate constants dropped more than 3000-fold as the volume of the E7 side chain, at position 64, was expanded from alanine (10(6) M-1 s-1) to phenylalanine (10(3) M-1 s-1). There was inhibition of imidazole binding due to displacement of coordinated water from H64 and H64Q sperm whale myoglobins, where the E7 side chain hydrogen bonds directly to the bound ligand. The imidazole dissociation rate constants varied less dramatically and less consistently with any single factor, though they were measurably decreased by hydrogen bonding to an E7 glutamine or histidine. On the whole, the results for the sperm whale myoglobin E7 substitutions show that the rate constants for imidazole binding are useful and sensitive indicators of steric hindrance and polar interactions in the distal pockets of myoglobins. The combined effects of the glutamine 64 and phenylalanine 29 in elephant myoglobin largely account for its increased imidazole association and dissociation rate constants, respectively, compared to those of sperm whale myoglobin. An unhindered distal pocket not competent to stabilize positive poles is indicated by the large imidazole association (>/=10(4) M-1 s-1) and dissociation (>/=50 s-1) rate constants, parameters that are characteristic of FixL.     

Determination of the affinity and kinetic constants for the interaction between the human virus echovirus 11 and its cellular receptor, CD55

The biochemical properties of the molecular interactions mediating viral-cell recognition are poorly characterized. In this study, we use surface plasmon resonance to study the affinity and kinetics of the interaction of echovirus 11 with its cellular receptor decay-accelerating factor (CD55). As reported for interactions between cell-cell recognition molecules, the interaction has a low affinity (KD approximately 3.0 microM) as a result of a very fast dissociation rate constant (kon approximately 10(5) M-1.s-1, koff approximately 0.3 s-1). This contrasts with the interaction of soluble ICAM-1 (sICAM-1, CD54) with human rhinovirus 3 which has been reported to have a similar affinity but 10(2)-10(3)-fold slower kinetics (Casasnovas, J. M., and Springer, T. A. (1995) J. Biol. Chem. 270, 13216-13224). The extracellular portion of decay-accelerating factor comprises four short consensus repeat domains (domains 1-4) and a mucin-like stalk. By comparison of the binding affinity for echovirus 11 of various fragments of decay-accelerating factor, we are able to conclude that short consensus repeat domain 3 contributes approximately 80% of the binding energy.     

Design of a ruthenium-cytochrome c derivative to measure electron transfer to the initial acceptor in cytochrome c oxidase

A ruthenium-labeled cytochrome c derivative was prepared to meet two design criteria: the ruthenium group must transfer an electron rapidly to the heme group, but not alter the interaction with cytochrome c oxidase. Site-directed mutagenesis was used to replace His39 on the backside of yeast C102T iso-1-cytochrome c with a cysteine residue, and the single sulfhydryl group was labeled with (4-bromomethyl-4' methylbipyridine) (bis-bipyridine)ruthenium(II) to form Ru-39-cytochrome c (cyt c). There is an efficient pathway for electron transfer from the ruthenium group to the heme group of Ru-39-cyt c comprising 13 covalent bonds and one hydrogen bond. Electron transfer from the excited state Ru(II*) to ferric heme c occurred with a rate constant of (6.0 +/- 2.0) x 10(5) s-1, followed by electron transfer from ferrous heme c to Ru(III) with a rate constant of (1.0 +/- 0.2) x 10(6) s-1. Laser excitation of a complex between Ru-39-cyt c and beef cytochrome c oxidase in low ionic strength buffer (5 mM phosphate, pH7) resulted in electron transfer from photoreduced heme c to CuA with a rate constant of (6 +/- 2) x 10(4) s-1, followed by electron transfer from CuA to heme a with a rate constant of (1.8 +/- 0.3) x 10(4) s-1. Increasing the ionic strength to 100 mM leads to bimolecular kinetics as the complex is dissociated. The second-order rate constant is (2.5 +/- 0.4) x 10(7) M-1s-1 at 230 mM ionic strength, nearly the same as that of wild-type iso-1-cytochrome c.     

Functional region IV of glycoprotein D from herpes simplex virus modulates glycoprotein binding to the herpesvirus entry mediator

Glycoprotein D (gD) of herpes simplex virus (HSV) is essential for virus entry and has four functional regions (I to IV) important for this process. We previously showed that a truncated form of a functional region IV variant, gD1(Delta290-299t), had an enhanced ability to block virus entry and to bind to the herpesvirus entry mediator (HveAt; formerly HVEMt), a cellular receptor for HSV. To explore this phenotype further, we examined other forms of gD, especially ones with mutations in region IV. Variant proteins with deletions of amino acids between 277 and 300 (region IV), as well as truncated forms lacking C-terminal residues up to amino acid 275 of gD, were able to block HSV entry into Vero cells 1 to 2 logs better than wild-type gD1(306t). In contrast, gD truncated at residue 234 did not block virus entry into Vero cells. Using optical biosensor technology, we recently showed that gD1(Delta290-299t) had a 100-fold-higher affinity for HveAt than gD1(306t) (3.3 x 10(-8) M versus 3.2 x 10(-6) M). Here we found that the affinities of other region IV variants for HveAt were similar to that of gD1(Delta290-299t). Thus, the affinity data follow the same hierarchy as the blocking data. In each case, the higher affinity was due primarily to a faster kon rather than to a slower koff. Therefore, once the gDt-HveAt complex formed, its stability was unaffected by mutations in or near region IV. gD truncated at residue 234 bound to HveAt with a lower affinity (2.0 x 10(-5) M) than did gD1(306t) due to a more rapid koff. These data suggest that residues between 234 and 275 are important for maintaining stability of the gDt-HveAt complex and that functional region IV is important for modulating the binding of gD to HveA. The binding properties of any gD1(234t)-receptor complex could account for the inability of this form of gDt to block HSV infection.     

Involvement of Neurospora mitochondrial tyrosyl-tRNA synthetase in RNA splicing. A new method for purifying the protein and characterization of physical and enzymatic properties pertinent to splicing

The Neurospora CYT-18 protein, the mitochondrial tyrosyl-tRNA synthetase, functions in the splicing of group I introns. Here, bacterially expressed CYT-18 protein, purified by a new procedure involving polyethyleneimine precipitation to remove tightly bound nucleic acids, was used to characterize properties pertinent to RNA splicing. Analytical ultracentrifugation and other methods showed that the CYT-18 protein is an asymmetric homodimer. The measured frictional ratio, f/fo = 1.55, corresponds to an axial ratio of 10 for a prolate ellipsoid or 12 for an oblate ellipsoid. Like bacterial TyrRSs, the CYT-18 protein exhibits half-sites reactivity, each homodimer having one active site for tyrosyl adenylation and RNA splicing. The splicing activity of CYT-18 was unaffected by aminoacylation substrates at concentrations used in aminoacylation reactions, whereas the TyrRS activity was inhibited by physiological concentrations of the splicing cofactor GTP, as well as CTP or UTP, or by low concentrations of a group I intron RNA. Kinetic measurements suggest that the binding of CYT-18 to a group I intron substrate is a two-step process, with an initial biomolecular step that is close to diffusion limited (3.24 +/- 0.03 x 10(7) M-1s-1) followed by a slower conformational change (0.54 +/- 0.07 s-1). After CYT-18 binding, splicing occurs at a rate of 0.0025 s-1, within 6-fold of the rate of self-splicing of the Tetrahymena large rRNA intron in vitro. The Kd for the complex between the CYT-18 protein and a group I intron substrate, calculated from koff/kon, was < 0.3 pM, substantially lower than determined by presumed equilibrium measurements [Guo, Q., & Lambowitz, A. M. (1992) Genes Dev. 6, 1357-1372]. As a result of this tight binding, the CYT-18 protein functions stoichiometrically in in vitro splicing reactions due to its extremely slow dissociation from the excised intron RNA. The very tight binding of the CYT-18 protein to the intron RNA raises the possibility that specific mechanisms exist for dissociating the protein from the excised intron in vivo.     

Kinetic properties of ba3 oxidase from Thermus thermophilus: effect of temperature

The kinetic properties of the ba3 oxidase from Thermus thermophilus were investigated by stopped-flow spectroscopy in the temperature range of 5-70 degrees C. Peculiar behavior in the reaction with physiological substrates and classical ligands (CO and CN-) was observed. In the O2 reaction, the decay of the F intermediate is significantly slower (k' = 100 s-1 at 5 degrees C) than in the mitochondrial enzyme, with an activation energy E of 10.1 +/- 0.9 kcal mol-1. The cyanide-inhibited ba3 oxidizes cyt c522 quickly (k approximately 5 x 10(6) M-1 s-1 at 25 degrees C) and selectively, with an activation energy E of 10.9 +/- 0.9 kcal mol-1, but slowly oxidizes ruthenium hexamine, a fast electron donor for the mitochondrial enzyme. Cyt c552 oxidase activity is enhanced up to 60 degrees C and is maximal at extremely low ionic strengths, excluding formation of a high-affinity cyt c522-ba3 electrostatic complex. The thermophilic oxidase is less sensitive to cyanide inhibition, although cyanide binding under turnover is much quicker (seconds) than in the fully oxidized state (days). Finally, the affinity of reduced ba3 for CO at 20 degrees C (Keq = 1 x 10(5) M-1) was found to be smaller than that of beef heart aa3 (Keq = 4 x 10(6) M-1), partly because of an unusually fast, strongly temperature-dependent CO dissociation from cyt a32+ of ba3 (k' = 0.8 s-1 vs k' = 0.02 s-1 for beef heart aa3 at 20 degrees C). The relevance of these results to adaptation of respiratory activity to high temperatures and low environmental O2 tensions is discussed.     

Calcium binding by the N-terminal cellulose-binding domain from Cellulomonas fimi beta-1,4-glucanase CenC

The interaction of the N-terminal cellulose-binding domain, CBDN1, from Cellulomonas fimi beta-1,4-glucanase CenC with calcium was investigated using NMR spectroscopy and calorimetry. CBDN1 binds a single calcium ion with an equilibrium association constant of approximately 10(5) M-1 at 35 degreesC and pH 6.0. Binding is exothermic (-42 +/- 2 kJ mol-1) under these conditions and is accompanied by a small negative change in heat capacity (DeltaCp = -0.41 +/- 0.16 kJ mol-1 K-1). From an NMR line shape analysis, the rate constants for calcium association and dissociation were found to be (5 +/- 2) x 10(7) s-1 M-1 and (4.5 +/- 0.6) x 10(2) s-1, respectively. The rapid association kinetics indicate that the calcium-binding site on CBDN1 is accessible and, to the first approximation, preformed. Based on patterns of chemical shift perturbations, and structural comparisons with the Bacillus sp. 1, 3-1,4-beta-glucanases, the backbone carbonyl oxygens of Thr8, Gly30, and Asp142 and a side chain carboxyl oxygen of Asp142 are postulated to form the calcium-binding site of CBDN1. Consistent with the calcium-independent affinity of CBDN1 for cellopentaose, this exposed site is located on the face of CBDN1 opposite to that forming the oligosaccharide-binding cleft. The midpoint denaturation temperature of CBDN1 is increased by approximately 8 degreesC at pH 6.0 in the presence of saturating amounts of calcium, confirming that metal ion binding is thermodynamically linked to native-state stability.     

Kinetic and spectroscopic investigations of wild-type and mutant forms of apple 1-aminocyclopropane-1-carboxylate synthase

Two catalytically inactive mutant forms of 1-aminocyclopropane-1-carboxylate (ACC) synthase, Y85A and K273A, were mixed in low concentrations of guanidine hydrochloride (GdnHCl). About 15% of the wild-type activity was recovered (theoretical 25% for a binomial distribution), proving that the functional unit of the enzyme is a dimer, or theoretically, a higher order oligomer. The enzyme catalyzes the conversion of S-adenosyl-L-methionine (SAM) to ACC. The value of kcat/KM is 1.2 x 10(6) M-1 s-1 at pH 8.3. Viscosity variation experiments with glycerol and sucrose as viscosogenic reagents showed that this reaction is nearly 100% diffusion controlled. The sensitivity to viscosity for the corresponding reaction of the less reactive Y233F mutant is much reduced, thus the latter reaction serves as a control for that of the wild-type enzyme. The kcat/KM vs pH profile for wild-type enzyme exhibits pKa values of 7.5 and 8.9. The former is assigned to the pKa of the alpha-amino group of SAM, while the latter corresponds to the independently determined spectrophotometric pKa of the internal aldimine. The kcat vs pH profile exhibits similar pKas, which means that the above pKa values are not perturbed in the Michaelis complex. The phenolic hydroxyl group of Tyr233 forms a hydrogen bond to the 3'-O- of PLP. The spectral and kinetic pKa (kcat/KM) values of the Y233F mutant are not identical (spectral 10.2, kinetic 8.7). A model that accounts quantitatively for these data posits two parallel pathways to the external aldimine for this mutant, the minor one has the alpha-amino group free base form of SAM reacting with the protonated imine form of the enzyme with kcat/KM approximately 6.0 x 10(3) M-1 s-1, while the major pathway involves reaction of the aldehyde form of PLP with SAM with kcat/KM approximately 7.0 x 10(5) M-1 s-1. The spectral pKa is defined only by the less reactive species.     

The heparan sulfate binding sequence of interferon-gamma increased the on rate of the interferon-gamma-interferon-gamma receptor complex formation

Interferon-gamma (IFNgamma), in common with a number of growth factors, binds both to heparan sulfate or heparin-related molecules and to a specific high affinity receptor (IFNgammaR). Using surface plasmon resonance technology, kinetic analysis of the IFNgamma. IFNgammaR complex formation was performed with the extracellular part of IFNgammaR immobilized on a sensor chip. At the sensor chip surface, IFNgamma was bound by two IFNgammaR molecules with an affinity in the nanomolar range (0.68 nM). This binding was characterized by an important on rate, kon = 7.3 x 10(6) M-1.s-1, and an off rate, koff = 5 x 10(-3).s-1. This binding assay was used to investigate a possible role of heparin in the IFNgamma.IFNgammaR complex formation. In contrast to growth factors for which binding to heparin is usually required for high affinity receptor interaction, we found in this study that IFNgamma bound to heparin displayed a strongly reduced affinity for its receptor. This is consistent with the fact that a cluster of basic amino acids (KTGKRKR, called the C1 domain) in the carboxyl-terminal sequence of the cytokine was involved both in heparin and receptor recognition. To understand how a single domain of IFNgamma could be implicated in two discrete functions (i.e. binding to heparin and to IFNgammaR), we also analyzed in a detailed manner the role of the IFNgamma carboxyl-terminal sequence in receptor binding. Using forms of IFNgamma, with carboxyl terminus truncations of defined regions of the heparin binding sequence, we found that the C1 domain functioned by increasing the on rate of the IFNgamma.IFNgammaR binding reaction but was not otherwise required for the stability of the complex. Interactions between the IFNgamma carboxyl-terminal domain and IFNgammaR could increased the association rate of the reaction either by increasing the number of encounters between the two molecules or by favoring productive collisions. The mechanisms by which heparan sulfate regulates IFNgamma activity may thus include both control of selective protease cleavage events, which directly affect the cytokine activity, and also an ability to modulate the interaction of IFNgamma with the IFNgammaR via competitive binding to the C1 domain.