NMR studies of the MgATP binding site of adenylate kinase and of a 45-residue peptide fragment of the enzyme

Proton NMR was used to study the interaction of beta,gamma-bidentate Cr3+ATP and MgATP with rabbit muscle adenylate kinase, which has 194 amino acids, and with a synthetic peptide consisting of residues 1-45 of the enzyme, which has previously been shown to bind MgepsilonATP [Hamada, M., Palmieri, R. H., Russell, G. A., & Kuby, S. A. (1979) Arch. Biochem. Biophys. 195, 155-177]. The peptide is globular and binds Cr3+ATP competitively with MgATP with a dissociation constant, KD(Cr3+ATP) = 35 microM, comparable to that of the complete enzyme [KI(Cr3+ATP) = 12 microM]. Time-dependent nuclear Overhauser effects (NOE's) were used to measure interproton distances on enzyme- and peptide-bound MgATP. The correlation time was measured directly for peptide-bound MgATP by studying the frequency dependence of the NOE's at 250 and 500 MHz. The H2' to H1' distance so obtained (3.07 A) was within the range established by X-ray and model-building studies of nucleotides (2.9 +/- 0.2 A). Interproton distances yielded conformations of enzyme- and peptide-bound MgATP with indistinguishable anti-glycosyl torsional angles (chi = 63 +/- 12 degrees) and 3'-endo/O1'-endo ribose puckers (sigma = 96 +/- 12 degrees). Enzyme- and peptide-bound MgATP molecules exhibited different C4'-C5' torsional angles (gamma) of 170 degrees and 50 degrees, respectively. Ten intermolecular NOE's from protons of the enzyme and four such NOE's from protons of the peptide to protons of bound MgATP were detected, which indicated proximity of the adenine ribose moiety to the same residues on both the enzyme and the peptide. Paramagnetic effects of beta,gamma-bidentate Cr3+ATP on the longitudinal relaxation rates of protons of the peptide provided a set of distances to the side chains of five residues, which allowed the location of the bound Cr3+ atom to be uniquely defined. Distances from enzyme-bound Cr3+ATP to the side chains of three residues of the protein agreed with those measured for the peptide. The mutual consistency of interproton and Cr3+ to proton distances obtained in metal-ATP complexes of both the enzyme and the peptide suggests that the conformation of the peptide is very similar to that of residues 1-45 of the enzyme. When this was assumed to be the case and when molecular models and a computer graphics system were used, MgATP could be fit into the X-ray structure of adenylate kinase in a unique manner such that all of the distances determined by NMR were accommodated.(ABSTRACT TRUNCATED AT 400 WORDS)     

Solution structure of the 45-residue MgATP-binding peptide of adenylate kinase as examined by 2-D NMR, FTIR, and CD spectroscopy

The structure of a synthetic peptide corresponding to residues 1-45 of rabbit muscle adenylate kinase has been studied in aqueous solution by two-dimensional NMR, FTIR, and CD spectroscopy. This peptide, which binds MgATP and is believed to represent most of the MgATP-binding site of the enzyme [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694], appears to maintain a conformation similar to that of residues 1-45 in the X-ray structure of intact porcine adenylate kinase [Sachsenheimer, W., & Schulz, G.E. (1977) J. Mol. Biol. 114, 23-26], with 42% of the residues of the peptide showing NOEs indicative of phi and psi angles corresponding to those found in the protein. The NMR studies suggest that the peptide is composed of two helical regions of residues 4-7 and 23-29, and three stretches of beta-strand at residues 8-15, 30-32, and 35-40, yielding an overall secondary structure consisting of 24% alpha-helix, 38% beta-structure, and 38% aperiodic. Although the resolution-enhanced amide I band of the peptide FTIR spectrum is broad and rather featureless, possibly due to disorder, it can be fit by using methods developed on well-characterized globular proteins. On this basis, the peptide consists of 35 +/- 10% beta-structure, 60 +/- 12% turns and aperiodic structure, and not more than 10% alpha-helix. The CD spectrum is best fit by assuming the presence of at most 13% alpha-helix in the peptide, 24 +/- 2% beta-structure, and 66 +/- 4% aperiodic. The inability of the high-frequency FTIR and CD methods to detect helices in the amount found by NMR may result from the short helical lengths as well as from static and dynamic disorder in the peptide. Upon binding of MgATP, numerous conformational changes in the backbone of the peptide are detected by NMR, with smaller alterations in the overall secondary structure as assessed by CD. Detailed assignments of resonances in the peptide spectrum and intermolecular NOEs between protons of bound MgATP and those of the peptide, as well as chemical shifts of peptide resonances induced by the binding of MgATP, are consistent with the previously proposed binding site for MgATP on adenylate kinase.     

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)     

Nuclear overhauser effect studies on the conformation of magnesium adenosine 5'-triphosphate bound to rabbit muscle creatine kinase

Nuclear Overhauser effects were used to determine interproton distances on MgATP bound to rabbit muscle creatine kinase. The internuclear distances were used in a distance geometry program that objectively determines both the conformation of the bound MgATP and its uniqueness. Two classes of structures were found that satisfied the measured interproton distances. Both classes had the same anti glycosidic torsional angle (chi = 78 +/- 10 degrees) but differed in their ribose ring puckers (O1'-endo or C4'-exo). The uniqueness of the glycosidic torsional angle is consistent with the preference of creatine kinase for adenine nucleotides. One of these conformations of MgATP bound to creatine kinase is indistinguishable from the conformation found for Co(NH3)4ATP bound to the catalytic subunit of protein kinase, which also has a high specificity for adenine nucleotides [chi = 78 +/- 10 degrees, O1'-endo; Rosevear, P.R., Bramson, H.N., O'Brian, C., Kaiser, E.T., & Mildvan, A.S. (1983) Biochemistry 22, 3439]. Distance geometry calculations also suggest that upper limit distances, when low enough (less than or equal to 3.4 A), can be used instead of measured distances to define, within experimental error, the glycosidic torsional angle of bound nucleotides. However, this approach does not permit an evaluation of the ribose ring pucker.     

Adenosine conformations of nucleotides bound to methionyl tRNA synthetase by transferred nuclear Overhauser effect spectroscopy.

The conformations of MgATP and AMP bound to a monomeric tryptic fragment of methionyl tRNA synthetase have been investigated by two-dimensional proton transferred nuclear Overhauser effect spectroscopy (TRNOESY). The sample protocol was chosen to minimize contributions from adventitious binding of the nucleotides to the observed NOE. The experiments were performed at 500 MHz on three different complexes, E.MgATP, E.MgATP.L-methioninol, and E.AMP.L-methioninol. A starter set of distances obtained by fitting NOE build-up curves (not involving H5' and H5") were used to determine a CHARMm energy-minimized structure. The positioning of the H5' and H5" protons was determined on the basis of a conformational search of the torsion angle to obtain the best fit with the observed NOEs for their superposed resonance. Using this structure, a relaxation matrix was set up to calculate theoretical build-up curves for all of the NOEs and compare them with the observed curves. The final structures deduced for the adenosine moieties in the three complexes are very similar, and are described by a glycosidic torsion angle (chi) of 56 degrees +/- 5 degrees and a phase angle of pseudorotation (P) in the range of 47 degrees to 52 degrees, describing a 3(4)T-4E sugar pucker. The glycosidic torsion angle, chi, deduced here for this adenylyl transfer enzyme and those determined previously for three phosphoryl transfer enzymes (creatine kinase, arginine kinase, and pyruvate kinase), and one pyrophosphoryl enzyme (PRibPP synthetase), are all in the range 52 degrees +/- 8 degrees. The narrow range of values suggests a possible common motif for the recognition and binding of the adenosine moiety at the active sites of ATP-utilizing enzymes, irrespective of the point of cleavage on the phosphate chain.     

Conformation of NADP+ bound to a type II dihydrofolate reductase

Type II dihydrofolate reductases (DHFRs) encoded by the R67 and R388 plasmids are sequence and structurally different from known chromosomal DHFRs. These plasmid-derived DHFRs are responsible for confering trimethoprim resistance to the host strain. A derivative of R388 DHFR, RBG200, has been cloned and its physical properties have been characterized. This enzyme has been shown to transfer the pro-R hydrogen of NADPH to its substrate, dihydrofolate, making it a member of the A-stereospecific class of dehydrogenases [Brito, R. M. M., Reddick, R., Bennett, G. N., Rudolph, F. B., & Rosevear, P. R. (1990) Biochemistry 29,9825]. Two distinct binary RBG200.NADP+ complexes were detected. Addition of NADP+ to RBG200 DHFR results in formation of an initial binary complex, conformation I, which slowly interconverts to a second more stable binary complex, conformation II. The binding of NADP+ to RBG200 DHFR in the second binary complex was found to be weak, KD = 1.9 +/- 0.4 mM. Transferred NOEs were used to determine the conformation of NADP+ bound to RBG200 DHFR. The initial slope of the NOE buildup curves, measured from the intensity of the cross-peaks as a function of the mixing time in NOESY spectra, allowed interproton distances on enzyme-bound NADP+ to be estimated. The experimentally measured distances were used to define upper and lower bound distance constraints between proton pairs in distance geometry calculations. All NADP+ structures consistent with the experimental distance bounds were found to have a syn conformation about the nicotinamide-ribose (X = 94 +/- 26 degrees) and an anti conformation about the adenine-ribose (X = -92 +/- 32 degrees) glycosidic bonds.(ABSTRACT TRUNCATED AT 250 WORDS)     

NMR studies of conformations and interactions of substrates and ribonucleotide templates bound to the large fragment of DNA polymerase I

The large fragment of DNA polymerase I (Pol I) effectively uses oligoribouridylates and oligoriboadenylates as templates, with kinetic properties similar to those of poly(U) and poly(A), respectively, and has little or no activity in degrading them. In the presence of such oligoribonucleotide templates, nuclear Overhauser effects (NOE's) were used to determine interproton distances within and conformations of substrates bound to the large fragment of Pol I, as well as conformations and interactions of the enzyme-bound templates. In the enzyme-oligo(rU)54 +/- 11-Mg2+dATP complex, the substrate dATP has a high anti-glycosidic torsional angle (chi = 62 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees) differing only slightly from those previously found for enzyme-bound dATP in the absence of template [Ferrin, L.J., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. Both conformations are similar to those of deoxynucleotidyl units of B DNA but differ greatly from those of A or Z DNA. The conformation of the enzyme-bound substrate analogue AMPCPP (chi = 50 +/- 10 degrees, delta = 90 +/- 10 degrees) is very similar to that of enzyme-bound dATP and is unaltered by the binding of the template oligo(rU)54 +/- 11 or by the subsequent binding of the primer (Ap)9A. In the enzyme-oligo(rA)50-Mg2+TTP complex, the substrate TTP has an anti-glycosidic torsional angle (chi = 40 +/- 10 degrees) and an O1'-endo sugar pucker (delta = 100 +/- 10 degrees), indistinguishable from those found in the absence of template and compatible with those of B DNA but not with those of A or Z DNA. In the absence of templates, the interproton distances on enzyme-bound dGTP cannot be fit by a single conformation but require a 40% contribution from a syn structure (chi = 222 degrees) and a 60% contribution from one or more anti structures. The presence of the template oligo(rU)43 +/- 9 simplifies the conformation of enzyme-bound dGTP to a single structure with an anti-glycosyl angle (chi = 32 +/- 10 degrees) and an O1'-endo/C3'-endo sugar pucker (delta = 90 +/- 10 degrees), compatible with those of B DNA, possibly due to the formation of a G-U wobble base pair. However, no significant misincorporation of guanine deoxynucleotides by the enzyme is detected with oligo(rU) as template.(ABSTRACT TRUNCATED AT 400 WORDS)     

In vitro mutagenesis studies at the arginine residues of adenylate kinase. A revised binding site for AMP in the X-ray-deduced model

Although X-ray crystallographic and NMR studies have been made on the adenylate kinases, the substrate-binding sites are not unequivocally established. In an attempt to shed light on the binding sites for MgATP2- and for AMP2- in human cytosolic adenylate kinase (EC 2.7.4.3, hAK1), we have investigated the enzymic effects of replacement of the arginine residues (R44, R132, R138, and R149), which had been assumed by Pai et al. [Pai, E. F., Sachsenheimer, W., Schirmer, R. H., & Schulz, G. E. (1977) J. Mol. Biol. 114, 37-45] to interact with the phosphoryl groups of AMP2- and MgATP2-. With use of the site-directed mutagenesis method, point mutations were made in the artificial gene for hAK1 [Kim, H. J., Nishikawa, S., Tanaka, T., Uesugi, S., Takenaka, H., Hamada, M., & Kuby, S. A. (1989) Protein Eng. 2, 379-386] to replace these arginine residues with alanyl residues and yield the mutants R44A hAK1, R132A hAK1, R138A hAK1, and R149A hAK1. The resulting large increases in the Km,app values for AMP2- of the mutant enzymes, the relatively small increases in the Km,app values for MgATP2-, and the fact that the R132A, R138A, and R149A mutant enzymes proved to be very poor catalysts are consistent with the idea that the assigned substrate binding sites of Pai et al. (1977) have been reversed and that their ATP-binding site may be assigned as the AMP site.     

Studies on the adenylate kinase isozymes from the serum and erythrocyte of normal and Duchenne dystrophic patients. Isolation, physicochemical properties, and several comparisons with the Duchenne dystrophic aberrant enzyme

Two species of adenylate kinase isozymes (ATP:AMP phosphotransferase, EC 2.7.4.3) from human Duchenne dystrophic serum were separated by Blue Sepharose CL-6B affinity column chromatography. One of these species was the "aberrant" adenylate kinase isozyme, found specifically in the Duchenne type of this disease (Hamada, M., Okuda, H., Oka, K., Watanabe, T., Ueda, K., Nojima, M., Kuby, S.A., Manship, M., Tyler, F., and Ziter, F. (1981) Biochim. Biophys. Acta 660, 227-237). The separated aberrant form possessed a molecular size of 98,000 (+/- 1,500), whereas the normal serum species of the enzyme was 87,000 (+/- 1,600) by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, by gel filtration, and by sedimentation equilibrium. The sedimentation coefficient of each species was found to be 5.8 S for the aberrant form and 5.6 S for the normal form, respectively. The subunit size (Mr = 24,700) of the aberrant enzyme in 8 M urea proved to be very similar to that of the normal human liver enzyme (Hamada, M., Sumida, M., Okuda, H., Watanabe, T., Nojima, M., and Kuby, S.A. (1982) J. Biol. Chem. 257, 13120-13128), and the normal species subunit (Mr = 21,700) was found to be very similar to that of the normal human muscle enzyme (Kuby, S.A., Fleming, G., Frischat, A., Cress, M.C., and Hamada, M. (1983) J. Biol. Chem. 258, 1901-1907). Both species were tetrameric enzymes in the serum. The amino acid composition for the normal species was similar to that for the muscle-type enzyme, and that for the aberrant species was similar to the liver enzyme, but with some notable exceptions in both cases. Thus, the normal species had no tryptophan and two half-cystine residues/subunit; whereas, there was 1 tryptophan and 4 half-cystine residues/subunit of the aberrant molecule. The amino acid composition of both serum isozymes when compared to their respective muscle or liver-type enzyme differed mainly in the content of Glu, Asp, His, Leu, Ile, Gly. Kinetic properties of the two forms of human serum adenylate kinase were studied at limiting concentrations of both ADP3- and MgADP- in the reverse reaction and of AMP2- and MgATP2- in the forward reaction. The type of reaction mechanism compatible with the data was a two-substrate random quasiequilibrium type of mechanism without independent binding of the substrates and with a rate-limiting step largely at the interconversion of the ternary complexes.     

Cloning, overexpression, and purification of aminoglycoside antibiotic nucleotidyltransferase (2'')-Ia: conformational studies with bound substrates

Aminoglycoside nucleotidyltransferase (2')-Ia [ANT (2')-Ia] was cloned from Pseudomonas aeruginosa and purified from overexpressing Escherichia coli BL21(DE3) cells. The first enzyme-bound conformation of an aminoglycoside antibiotic in the active site of an aminoglycoside nucleotidyltransferase was determined using the purified aminoglycoside nucleotidyltransferase (2' ')-Ia. The conformation of the aminoglycoside antibiotic isepamicin, a psuedo-trisaccharide, bound to aminoglycoside nucleotidyltransferase (2' ')-Ia has been determined using NMR spectroscopy. Molecular modeling, employing experimentally determined interproton distances, resulted in two different enzyme-bound conformations (conformer 1 and conformer 2) of isepamicin. Conformer 1 was by far the major conformer defined by the following average glycosidic dihedral angles: PhiBC = -65.26 +/- 1.63 degrees and PsiBC = -54.76 +/- 4.64 degrees. Conformer 1 was further subdivided into one major (conformer 1a) and two minor components (conformers 1b and 1c) based on the comparison of glycosidic dihedral angles PhiAB and PsiAB. The arrangement of substrates in the enzyme.metal-ATP.isepamicin complex was determined on the basis of the measured effect of the paramagnetic substrate analogue Cr(H2O)4ATP on the relaxation rates of substrate protons which were used to determine relative distances of isepamicin protons to the Cr3+. Both conformers of isepamicin yielded arrangements that satisfied the NOE restraints and the observed paramagnetic effects of Cr(H2O)4ATP. It has been suggested that aminoglycosides use both electrostatic interactions and hydrogen bonds in binding to RNA and that the contacts made by the A and B rings to RNA are the most important for binding [Fourmy, D., Recht, M. I., Blanchard, S. C., and Puglisi, J. D. (1996) Science 274, 1367-1371]. Comparisons based on the determined conformations of enzyme-bound aminoglycoside antibiotics also suggested that interactions of rings A and B with enzymes may be the major determinant in aminoglycoside binding to enzymes [Serpersu, E. H., Cox, J. R., DiGiammarino, E. L., Mohler, M. L., Ekman, D. R., Akal-Strader, A., and Owston, M. (2000) Cell Biochem. Biophys. (in press)]. The conformation of isepamicin bound to the aminoglycoside nucleotidyltransferase (2' ')-Ia, determined in this work, lent further support to this theory. Furthermore, comparison of enzyme-bound conformations of isepamicin to the RNA-bound conformation of gentamycin C1a also showed remarkable similarities between the enzyme-bound and RNA-bound aminoglycoside antibiotic conformations. These studies should aid in the design of effective inhibitors possessing a broad range of aminoglycoside-modifying enzymes as targets.     

NMR studies of the backbone protons and secondary structure of pentapeptide and heptapeptide substrates bound to bovine heart protein kinase

The conformations of enzyme-bound pentapeptide (Arg-Arg-Ala-Ser-Leu) and heptapeptide (Leu-Arg-Arg-Ala-Ser-Leu-Gly) substrates of protein kinase have been studied by NMR in quaternary complexes of the type (Formula: see text). Paramagnetic effects of Mn2+ bound at the inhibitory site of the catalytic subunit on the longitudinal relaxation rates of backbone Ca protons, as well as on side-chain protons of the bound pentapeptide and heptapeptide substrates, have been used to determine Mn2+ to proton distances which range from 8.2 to 12.4 A. A combination of the paramagnetic probe-T1 method with the Redfield 2-1-4-1-2 pulse sequence for suppression of the water signal has been used to measure distances from Mn2+ to all of the backbone amide (NH) protons of the bound pentapeptide and heptapeptide substrates, which range from 6.8 to 11.1 A. Paramagnetic effects on the transverse relaxation rates yield rate constants for peptide exchange, indicating that the complexes studied by NMR dissociate rapidly enough to participate in catalysis. Model-building studies based on the Mn2+-proton distances, as well as on previously determined distances from Cr3+-AMPPCP to side-chain protons [Granot, J., Mildvan, A.S., Bramson, H. N., & Kaiser, E. T. (1981) Biochemistry 20, 602], rule out alpha-helical, beta-sheet, beta-bulge, and all possible beta-turn conformations within the bound pentapeptide and heptapeptide substrates. The distances are fit only by extended coil conformations for the bound peptide substrates with a minor difference between the pentapeptides and heptapeptides in the phi torsional angle at Arg3C alpha and in psi at Arg2C alpha. An extended coil conformation, which minimizes the number of interactions within the substrate, would facilitate enzyme-substrate interaction and could thereby contribute to the specificity of protein kinase.     

Mechanism of adenylate kinase. Demonstration of a functional relationship between aspartate 93 and Mg2+ by site-directed mutagenesis and proton, phosphorus-31, and magnesium-25 NMR

Earlier magnetic resonance studies suggested no direct interaction between Mg2+ ions and adenylate kinase (AK) in the AK.MgATP (adenosine 5'-triphosphate) complex. However, recent NMR studies concluded that the carboxylate of aspartate 119 accepts a hydrogen bond from a water ligand of the bound Mg2+ ion in the muscle AK.MgATP complex [Fry, D.C., Kuby, S.A., & Mildvan, A.S. (1985) Biochemistry 24, 4680-4694]. On the other hand, in the 2.6-A crystal structure of the yeast AK.MgAP5A [P1,P5-bis(5'-adenosyl)pentaphosphate] complex, the Mg2+ ion is in proximity to aspartate 93 [Egner, U., Tomasselli, A.G., & Schulz, G.E. (1987) J. Mol. Biol. 195, 649-658]. Substitution of Asp-93 with alanine resulted in no change in dissociation constants, 4-fold increases in Km, and a 650-fold decrease in kcat. Notable changes have been observed in the chemical shifts of the aromatic protons of histidine 36 and a few other aromatic residues. However, the results of detailed analyses of the free enzymes and the AK.MgAP5A complexes by one- and two-dimensional NMR suggested that the changes are due to localized perturbations. Thus it is concluded that Asp-93 stabilizes the transition state by ca. 3.9 kcal/mol. The next question is how. Since proton NMR results indicated that binding of Mg2+ to the AK.AP5A complex induces some changes in the proton NMR signals of WT but not those of D93A, the functional role of Asp-93 should be in binding to Mg2+.(ABSTRACT TRUNCATED AT 250 WORDS)     

Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 1. Adenosine conformations by proton two-dimensional transferred nuclear Overhauser effect spectroscopy

Adenosine conformations of adenosine 5'-triphosphate (ATP) and adenosine 5'-monophosphate (AMP), and of an ATP analogue, adenylyl imidodiphosphate (AMPPNP), bound to Escherichia coliadenylate kinase (AKe) in the complexes of AKe.Mg(II)ATP, AKe.AMP.Mg(II)GDP, AKe. AMPPNP, and AKe.Mg(II)AMPPNP were determined by transferred two-dimensional nuclear Overhauser effect spectroscopy (TRNOESY) measurements and molecular dynamics simulations. The glycosidic torsion angles, chi, deduced for the adenine nucleotides in these complexes are 51 degrees, 37 degrees, 49 degrees, and 47 degrees, respectively, with an experimental error of about +/-5 degrees. These values are in general agreement with those previously measured for other ATP-utilizing enzymes, suggesting a possible common motif for adenosine recognition and binding. The pseudorotational phase angle, P, of the sugar puckers for the bound nucleotides varied between 50 degrees and 103 degrees. These solution-state conformations are significantly different from those in published data from X-ray crystallography. A computation of the ligand NOEs, made by using the program CORCEMA [Moseley, H. N. B., Curto, E. V., and Krishna, N. R. (1995) J. Magn. Reson. B108, 243-261] with the protein protons in the vicinity of nucleotide included, on the basis of the X-ray structure of the AKe.AMP.AMPPNP complex [Berry, M. B., Meador, B., Bilderback, T., Liang, P., Glaser, M., and Philips, G. N. , Jr. (1994) Proteins: Struct., Funct., Genet. 19, 183-198], showed that polarization transfer to the protein protons does not produce significant errors in the structures determined by considering the ligand NOEs alone.     

Structural characterization of adenine nucleotides bound to Escherichia coli adenylate kinase. 2. 31P and 13C relaxation measurements in the presence of cobalt(II) and manganese(II)

13C spin-lattice relaxation rates have been measured for two complexes of Escherichia coli adenylate kinase (AKe), viz., AKe. [U-(13)C]ATP and AKe.[U-(13)C]AMP.GDP in the presence of the substituent activating paramagnetic cation Mn(II) for the purpose of determination of the enzyme-bound conformations of ATP and AMP. (GDP has been added to the AMP complex with the enzyme in order to hold the cation in the bound complex.) Measurements of relaxation times at three different (13)C frequencies, 181.0, 125.7, and 75.4 MHz, indicate that the relaxation times in the enzyme-nucleotide complexes with the paramagnetic cation are not exchange-limited; i.e. , they are larger than the effective lifetimes of cation binding to these complexes and are, therefore, dependent on the cation-(13)C distances. An analysis of the frequency-dependent relaxation data allowed all of the ten Mn(II)-(13)C distances to be determined in each of the complexes. Similar measurements of the (31)P relaxation rate made on AKe.ATP and AKe.AMP.GDP complexes in the presence of Co(II) as the activating cation yielded Co(II)-(31)P distances for each adenine nucleotide. These distances, together with the interproton distances determined previously from TRNOESY experiments [Lin, Y., and Nageswara Rao, B. D. (2000) Biochemistry 39, 3636-3646], led to a complete characterization of both ATP and AMP conformations in AKe-bound complexes. These conformations differ significantly from the nucleotide conformations in crystals of AKe. AP(5)A and AKe.AMP.AMPPNP as determined by X-ray crystallography.     

Conformations and arrangement of substrates at active sites of ATP-utilizing enzymes

The phosphoryl transferring enzymes pyruvate kinase, cAMP-dependent protein kinase and the pyrophosphoryl transferring enzyme PP-Rib-P synthetase utilize the beta, gamma bidentate metal--ATP chelate (delta-isomer) as substrate, as determined with substitution-insert CrIIIATP or CoIII(NH3)4ATP complexes. In addition, these enzymes bind a second divalent cation, which is an essential activator for pyruvate kinase and PP-Rib-P synthetase and an inhibitor of protein kinase. The enzyme-bound metal has been used as a paramagnetic reference point in T1 measurements to determine distances to the protons and phosphorus atoms of the bound nucleotide and acceptor substrates. These distances have been used to construct models of the conformations of the bound substrates. The activating metal forms a second sphere complex of the metal-nucleotide substrate on pyruvate kinase and PP-Rib-P synthetase while the inhibitory metal directly coordinates the polyphosphate chain of the metal-nucleotide substrate on protein kinase. Essentially no change is found in the dihedral angle at the glycosidic bond of ATP upon binding to pyruvate kinase (chi = 30 degrees), an enzyme of low base specificity, but significant changes in the torsional angle of ATP occur on binding to protein kinase (chi = 84 degrees) and PP-Rib-P synthetase (chi = 62 degrees), enzymes with high adenine-base specificity. Intersubstrate distances, measured with tridentate CrATP or beta, gamma bidentate CrAMPPCP as paramagnetic reference points, have been used to deduce the distance along the reaction coordinate on each enzyme. The reaction coordinate distances on pyruvate kinase (# +/- 1 A) and PP-Rib-P synthetase (not less than 3.8 A) are consistent with associative mechanisms, while that on protein kinase (5 +/- 0.7 A) allows room for a dissociative mechanism.     

The steady-state kinetics of the enzyme reaction tested by site-directed mutagenesis of hydrophobic residues (Val, Leu, and Cys) in the C-terminal alpha-helix of human adenylate kinase

To elucidate whether the C-terminal region in human adenylate kinase participates in the interaction with the substrate (MgATP(2-) and/or AMP(2-)), hydrophobic residues (Val182, Val186, Cys187, Leu190, and Leu193) were substituted by site-directed mutagenesis and the steady-state kinetics of fifteen mutants were analyzed. A change in the hydrophobic residues in the C-terminal domain affects the affinity for substrates (K(m)), that is, not only for MgATP(2-) but also for AMP(2-), and the catalytic efficiency (k(cat)). The results obtained have led to the following conclusions: (i) Val182 may interact with both MgATP(2-) and AMP(2-) substrates, but to a greater extent with MgATP(2-), and play a role in catalysis. (ii) Val186 appears to play a functional role in catalysis by interacting with both MgATP(2-) and AMP(2-) to nearly the same extent. (iii) Cys187 appears to play a functional role in catalysis. (iv) Leu190 appears to interact with both MgATP(2-) and AMP(2-) substrates but to a greater extent with AMP(2-). (v) Leu193 appears to interact with both MgATP(2-) and AMP(2-) but to a greater extent with AMP(2-). The activity of all mutants decreased due to the change in substrate-affinity. The closer the residue is located to the C-terminal end, the more its mutation affects not only MgATP(2-) but also AMP(2-) substrate binding. The hydrophobic alterations disrupt hydrophobic interactions with substrates and that might destabilize the conformation of the active site. The more C-terminal part of the alpha-helix appears to interact with AMP, as if it has swung out and rotated to cover the adenine moieties. The C-terminal alpha-helix of human adenylate kinase appears to be essential for the interaction with adenine substrates by swinging out during catalysis.     

8-Azido-2'-O-dansyl-ATP. A fluorescent photoaffinity reagent for ATP-binding proteins and its application to adenylate kinase

The photoaffinity reagent 8-azido-2'-O-[14C]dansyl-ATP (AD-ATP) has been synthesized for labeling and monitoring the active sites of ATPases and kinases. In its first application, the reagent is used to explore the active site of adenylate kinase from rabbit muscle. In the dark, AD-ATP inhibits adenylate kinase reversibly and competitively with KI = 0.25 +/- 0.01 microM. Under weak UV illumination, AD-ATP labels adenylate kinase irreversibly. The photoinactivation data also show KI = 0.25 +/- 0.02 microM. The ratio (r) of the specific activity of AD-ATP-labeled adenylate kinase to that of the unlabeled enzyme has been determined as a function of the number (n) of label/enzyme. The linear plot of r versus n with slope equal to -1 shows that the labeling is very specific, i.e. each label completely inactivates an enzyme molecule. After the labeled enzyme was partially hydrolyzed and the radioactive peptides analyzed and sequenced, it was found that Leu-115, Cys-25, and probably His-36 were labeled, in agreement with previous conclusions on the structure of the active site of this enzyme based on amino acid sequence, x-ray diffraction, and NMR studies. The environment-sensitive fluorescent dansyl group of AD-ATP can function as an in situ probe for monitoring ligand or conformation changes at the active site. The fluorescence of AD-ATP-labeled enzyme with n = 0.9 is not affected by ATP but increases with the concentration of AMP in solution. This observation is also in agreement with the previous conclusion that ATP does not bind to the AMP site of adenylate kinase. The observed enhancement of fluorescence indicates that binding of AMP by this enzyme causes environmental change at its ATP site. The possible usefulness of AD-ATP as an effective biological inhibitor or as a molecular probe for studying the structure and regulation of ATP-binding proteins is discussed.     

Uniform 15N labeling of a fungal peptide: the structure and dynamics of an alamethicin by 15N and 1H NMR spectroscopy.

An alamethicin, secreted by the fungus Trichoderma viride and containing a glutamine at position 18 instead of the usual glutamic acid, has been uniformly labeled with 15N and purified by HPLC. The extent of 15N incorporation at individual backbone and side-chain sites was found to vary from 85% to 92%, as measured by spin-echo difference spectroscopy. The proton NMR spectrum of the peptide dissolved in methanol was assigned using correlation spectroscopies and nuclear Overhauser enhancements (NOE) measured in the rotating frame. The 15N resonances were assigned by the 2D 1H-15N correlation via heteronuclear multiple-quantum coherence experiment. NOEs and 3JNHC alpha H coupling constants strongly suggest that, in methanol, from Aib-3 to Gly-11, the peptide adopts a predominantly helical conformation, in agreement with previous 1H NMR studies [Esposito, G., Carver, J.A, Boyd, J., & Campbell, I.D. (1987) Biochemistry 26, 1043-1050; Banerjee, U., Tsui, F.-P., Balasubramanian, T.N., Marshall, G.R., & Chan, S I. (1983) J. Mol. Biol. 165, 757-775]. The conformation of the carboxyl terminus (12-20) is less well determined, partly because the amino acid composition reduces the number of NOEs and coupling constants which can be determined by 1H NMR spectroscopy. The 3JNHC alpha H in the C-terminus suggest the possibility of conformational averaging at Leu-12, Val-15, and Gln-19, an interpretation which is supported by a recent molecular dynamics simulation of the peptide [Fraternalli, F. (1990) Biopolymers 30, 1083-1099].(ABSTRACT TRUNCATED AT 250 WORDS)     

Conformation of MgATP bound to nucleotidyl and phosphoryl transfer enzymes 1H-transferred NOE measurements on complexes of methionyl tRNA synthetase and pyruvate kinase.

The conformations of MgATP bound to a nucleotidyl transfer enzyme, methionyl tRNA synthetase and a phosphoryl transfer enzyme, pyruvate kinase, were studied by transferred NOE (TRNOE) measurements in 1H NMR. The experiments were performed on D2O solutions at 276 MHz and 300 MHz, and 10 degrees C in the presence of approximately a tenfold excess of substrate over the enzyme (sites). Selective inversion of chosen resonances was accomplished with an appropriately tailored DANTE sequence consisting of 100 phase-alternating hard 1.8 degree pulses. NOE measurements were made in terms of difference spectra (with and without inversion) at 6-8 delay times ranging from 10-500 ms following the DANTE sequence. A full complement of ten NOE build-up curves obtained for each enzyme complex was analyzed by using the complete relaxation-matrix method (which includes all the non-exchangeable protons in MgATP) suitably modified to include exchange between bound and free substrate. Molecular mechanics computations were used to examine the energetic implications of the NOE-determined structure. The final structures obtained for MgATP bound to the two enzymes were very similar to each other, with a 3'-endo sugar pucker and an anti conformation with a glycosidic torsional angle (O'4-C'1-N9-C8) of 39 degrees +/- 4 degrees. Both enzymes contain multiple binding sites for MgATP and hence the structure obtained in each case represents an average due to chemical exchange. However, TRNOE experiments performed on a tryptic fragment of methionyl tRNA synthetase which has a single MgATP binding site, show that the same structure fits these measurements as well. This evidence, coupled with the striking similarity of the structures deduced, for the two enzyme complexes, and the reciprocal sixth-power dependence of NOE on interproton distance, strongly suggests that the conformations at the individual binding sites of both the enzymes are virtually identical. This conclusion is in contrast with multiple conformations of MgATP bound to pyruvate kinase, proposed by Rosevear, P.R., Fox, T.L. & Mildvan, A.S. (1987) Biochemistry 26, 3487-3493.     

Cloning,overexpression, and purification of aminoglycoside antibiotic 3-acetyltransferase-IIIb: conformational studies with bound substrates

Aminoglycoside 3-acetyltransferase-IIIb (AAC3), which acetylates N3 amine of aminoglycoside antibiotics, was cloned from P. Aeruginosa and purified from overexpressing E. coli BL21 (DE3) cells. Bound conformations of kanamycin A and ribostamycin, in the active site of the enzyme that modifies the essential N3B of aminoglycoside antibiotics, were determined by NMR spectroscopy. Experimentally determined interproton distances were used in a simulated annealing protocol to determine enzyme-bound conformations of both antibiotics. Two conformations, consistent with the NOE restraints, were determined for ribostamycin. The only difference between the two conformers was the orientation of the A ring with respect to the rest of the molecule. The average glycosidic dihedral angles were Phi(1A) = -22 degrees +/- 3 and Psi(1A) = -42 degrees +/- 1 (conformer 1) and Phi(1A) = -67 degrees +/- 0.7 and Phi(1A) = -59 degrees +/- 0.8 (conformer 2). Three conformers were determined for the enzyme-bound kanamycin A. Two conformers of kanamycin A were matched well with the two conformers of ribostamycin when the A and the B rings of the antibiotics were superimposed. Conformations of kanamycin A and ribostamycin were compared to those of other aminoglycosides that are bound to different enzymes and RNA. The results lend further support to our earlier hypothesis that the A and B rings of aminoglycosides adopt a conformation that is recognized not only by the aminoglycoside-modifying enzymes but also by RNA (Serpersu, E. H., Cox, J. R., Digiammarino, E. L., Mohler, M. L., Akal, A., Ekman, D. R., and Owston, M. (2000) Cell Biochem. Biophys. 33, 309-321). These results may be useful in designing new antibiotics to combat the antibiotic resistance against infectious diseases.