Hydrogen exchange kinetics of bovine pancreatic trypsin inhibitor beta-sheet protons in trypsin-bovine pancreatic trypsin inhibitor, trypsinogen-bovine pancreatic trypsin inhibitor, and trypsinogen-isoleucylvaline-bovine pancreatic trypsin inhibitor

Hydrogen exchange rates of six beta-sheet peptide amide protons in bovine pancreatic trypsin inhibitor (BPTI) have been measured in free BPTI and in the complexes trypsinogen-BPTI, trypsinogen-Ile-Val-BPTI, bovine trypsin-BPTI, and porcine trypsin-BPTI. Exchange rates in the complexes are slower for Ile-18, Arg-20, Gln-31, Phe-33, Tyr-35, and Phe-45 NH, but the magnitude of the effect is highly variable. The ratio of the exchange rate constant in free BPTI to the exchange rate constant in the complex, k/kcpIx, ranges from 3 to much greater than 10(3). Gln-31, Phe-45, and Phe-33 NH exchange rate constants are the same in each of the complexes. For Ile-18 and Tyr-35, k/kcpIx is much greater than 10(3) for the trypsin complexes but is in the range 14-43 for the trypsinogen complexes. Only the Arg-20 NH exchange rate shows significant differences between trypsinogen-BPTI and trypsinogen-Ile-Val-BPTI and between porcine and bovine trypsin-BPTI.     

Comparison of hydrogen exchange rates for bovine pancreatic trypsin inhibitor in crystals and in solution.

Hydrogen exchange rate constants for the 17 slowest exchanging amide NH groups in bovine pancreatic trypsin inhibitor (BPTI) were measured in solution and in form II and form III crystals. All 17 amide hydrogens are buried and intramolecularly hydrogen bonded in the crystal structure, except Lys 41 which is buried and hydrogen bonded to a buried water. Large-scale crystallization procedures were developed for these experiments, and rate constants for both crystal and solution exchange were measured by 1H NMR spectroscopy of exchange-quenched samples in solution. Two conditions of pH and temperature, pH 9.8 and 35 degrees C, and pH 9.4 and 25 degrees C, bring two groups of hydrogens into the experimental time window (minutes to weeks). One consists of the 10 slowest exchanging hydrogens, all of which are associated with the central beta-sheet of BPTI. The second group consists of seven more rapidly exchanging hydrogens, which are distributed throughout the molecule, primarily in a loop or turn. In both groups, most hydrogens exchange more slowly in crystals, but there is considerable variation in the degree to which the exchange is depressed in crystals. Many differences observed for the more rapidly exchanging hydrogens can be attributed to local surface effects arising from intermolecular contacts in the crystal lattice. Within the slower group, however, a very large effect on exchange of Ile 18 and Tyr 35 appears to be selectively transmitted through the matrix of the molecule.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accelerated exchange of a buried water molecule in selectively disulfide-reduced bovine pancreatic trypsin inhibitor

Using magnetic relaxation dispersion (MRD), we have previously shown that the four internal water molecules in bovine pancreatic trypsin inhibitor (BPTI) exchange with bulk water on time scales between 10(-8) and 10(-4) s at room temperature. Because this exchange is controlled by the protein structure, internal water molecules can be used to probe rare conformational fluctuations. Here, we report (2)H and (17)O MRD data at three temperatures for wild-type BPTI and two BPTI variants where the 14-38 disulfide bond has been cleaved by a double Cys --> Ser mutation or by disulfide reduction and carboxamidomethylation. The MRD data show that the internal water molecules are conserved on disulfide cleavage. However, the exchange rate of the water molecule buried near the disulfide bond is enhanced by 2-4 orders of magnitude. The relation of water exchange to other dynamic processes in BPTI is discussed.     

Hydrogen exchange of primary amide protons in basic pancreatic trypsin inhibitor: evidence for NH2 group rotation in buried asparagine side chains

Hydrogen-deuterium exchange is measured for the buried primary amide groups of Asn-43 and Asn-44 in bovine pancreatic trypsin inhibitor. Amide protons trans and cis to the amide carbonyl oxygen (HE and HZ, respectively) exchange at indistinguishable rates. Uncorrelated exchange of HE and HZ is established for both residues by following the nuclear Overhauser enhancement from HE to HZ during the deuterium exchange. The exchange of Asn-43 and Asn-44 side-chain protons differs qualitatively from exchange of primary amide groups in fully solvated model compounds, for which HE generally exchanges faster than HZ. The equal rates for the buried primary amide HE and HZ in BPTI are not a consequence of coupled exchange. The data indicate rapid rotation around the CO-NH2 bond for both Asn-43 and Asn-44 and suggest considerable lability of intramolecular hydrogen bonds. The side chain of Asn-43 has all of its polar atoms integrated into the very stable hydrogen-bonded structure of the protein. Asn-44 is hydrogen-bonded to side chains and to a buried water molecule. Solvent isotope exchange is several orders of magnitude more restricted by protein secondary and tertiary structure than the CO-NH2 rotation, indicating that N delta H2 groups flip many times before hydrogen isotope exchange occurs.     

Correlation between calculated local stability and hydrogen exchange rates in proteins

The attempt is made to find new correlations between local structural characteristics of proteins and the hydrogen exchange rates of their individual main-chain amides, and to relate such correlations to possible mechanisms of hydrogen exchange. It is found that in bovine pancreatic trypsin inhibitor (BPTI) the surface area buried by a particular residue and its neighbors correlates with the exchange rate of the main-chain amide of that residue. As the area buried by a particular fragment can be associated with the stabilization of the protein structure by this fragment, the correlation suggests a role for the energetics of the local unfolding in the mechanism of hydrogen exchange. Calculations based on the assumption that the exchange mechanism involves local unfolding lead to quantitative agreement between the calculated and experimentally measured exchange rates for 80% of the amides of BPTI that are buried or hydrogen bonded to the main-chain or to internal water molecules. The same degree of correlation is found between the calculated exchange rates and partial exchange data for ribonuclease S, hen lysozyme and cytochrome c. A similarly strong correlation is found between calculated exchange rates and the exchange rates of ribonuclease A determined by neutron diffraction in the crystal. The criteria of correlation are, however, less stringent in this case because of the experimental errors, which are larger than for solution data. It is suggested that the observed correlation be used for predictions of hydrogen exchange rates in proteins.     

Mechanism of surface peptide proton exchange in bovine pancreatic trypsin inhibitor. Salt effects and O-protonation

The acid-catalyzed hydrogen exchange rate constants kH, and the base-catalyzed rate constants kOH, have been determined (in the preceding paper) for the 25 most rapidly exchanging NH groups of bovine pancreatic trypsin inhibitor. Most of these NH groups are at the protein-solvent interface. The correlation of kH, but not kOH, with the static accessibility and hydrogen bonding of the peptide carbonyl O atom indicates that the mechanism of acid catalysis in proteins involves O-protonation. Agreement between the ionic strength dependence observed for kH and kOH and the ionic strength dependence calculated for an O-protonation mechanism supports this conclusion. N-protonation for acid catalysis, as well as N-deprotonation for base catalysis, have traditionally been assumed in the mechanism of the chemical step in peptide amide proton exchange. A preference for the alternative O-protonation mechanism has far-reaching implications in the interpretation of protein hydrogen exchange kinetics. With an O-protonation mechanism, acid-catalyzed rates of surface NH groups are primarily a function of the average solvent accessibility of the carbonyl O atoms in the dynamic solution structure, while base-catalyzed rates of surface NH groups measure solvent accessibility of the peptide N. The relative dynamic accessibilities of peptide O atoms, as measured by relative values of kH (corrected for electrostatic effects), correlate with O static accessibilities in the crystal structure. A lower correlation of static accessibility of N atoms with kOH is observed for surface NH groups in peptide groups in which the carbonyl O is not hydrogen bonded. For some surface NH groups, the observed pH of minimum rate, pHmin, deviates widely from the pHmin of model compounds. This is explained as the combined result of electrostatic effects and of the differences in accessibility of the carbonyl O and N atoms that result in a change in the relative values of kH and kOH as compared to those of model peptides. A mechanism whereby exchange of interior sites is catalyzed by interactions of catalysis ions with protein surface atoms via charge transfer is suggested.     

Hydrogen bonding monitored by deuterium isotope effects on carbonyl 13C chemical shift in BPTI: intra-residue hydrogen bonds in antiparallel beta-sheet.

Deuterium isotope effects on carbonyl 13C magnetic shielding were measured for the backbone carbonyl groups in BPTI (basic pancreatic trypsin inhibitor), and interpreted as a measure of hydrogen bond energies. The effects originate from peptide amide proton deuterium substitution and were observed on carbonyl carbons separated by two or three covalent bonds from the amide H/D. Two-bond isotope effects depend on the energy of the hydrogen bond donated by NH/D. Calibration of the effect with model compound data leads to hydrogen bond enthalpies less than 4.7 kcal/mol. Isotope effects over three bonds from the amide H/D to the carbonyl carbon of the same amino acid residue are observed for seven carbonyl resonances in BPTI. The three-bond isotope effects are highly related to the various backbone conformations. The largest effects are observed for residues with an approximate syn- periplanar conformation of the H-N-C alpha-C = O atoms, as realized for many residues in the BPTI antiparallel beta-sheet. The residues that show measurable three-bond effects have unusually short distances between H and O. The size of this effect decreases rapidly with increased O..H distance in the open five-membered ring. This observation suggests appreciable interactions in these rings.     

On the pH dependence of amide proton exchange rates in proteins.

We have analyzed the pH dependencies of published amide proton exchange rates (kex) in three proteins: bovine pancreatic trypsin inhibitor (BPTI), bull seminal plasma proteinase inhibitor IIA (BUSI IIA), and calbindin D9K. The base-catalyzed exchange rate constants (kOH) of solvent exposed amides in BPTI are lower for residues with low peptide carbonyl exposure, showing that the environment around the carbonyl oxygen influences kOH. We also examined the possible importance of an exchange mechanism that involves formations of imidic acid intermediates along chains of hydrogen-bonded peptides in the three proteins. By invoking this "relayed imidic acid exchange mechanism," which should be essentially acid-catalyzed, we can explain the surprisingly high pHmin (the pH value at which kex reaches a minimum) found for the non-hydrogen-bonded amide protons in the beta-sheet in BPTI. The successive increase of pHmin along a chain of hydrogen-bonded peptides from the free amide to the free carbonyl, observed in BPTI, can be explained as an increasing contribution of the proposed mechanism in this direction of the chain. For BUSI IIA (pH 4-5) and calbindin D9K (pH 6-7) the majority of amide protons with negative pH dependence of kex are located in chains of hydrogen-bonded peptides; this situation is shown to be consistent with the proposed mechanism.     

Mechanistic investigations of Escherichia coli cytidine-5'-triphosphate synthetase. Detection of an intermediate by positional isotope exchange experiments

CTP synthetase from Escherichia coli catalyzes exchange of 18O from the beta gamma-bridge position of [gamma-18O4] ATP into the beta-nonbridge position. This positional isotope exchange occurs in the presence of UTP and MgCl2 but in the absence of NH3. The enzyme also has an ATPase activity in the presence of UTP that occurs under conditions that are identical to those used in the positional isotope exchange experiments. These data provide evidence for the stepwise nature of the reactions catalyzed by CTP synthetase with the initial step involving phosphorylation of UTP by ATP. The relative rate of the isotope exchange reaction is approximately 3 times faster than the ATPase reaction, but the isotope exchange rate is approximately 3% of the overall rate in the presence of NH3. These results are consistent with the ATPase reaction involving attack of water on the phosphorylated intermediate (4-phospho-UTP). The positional isotope exchange reaction is independent of the UTP concentration above saturating levels of UTP demonstrating that the order of addition of substrates is UTP followed by ATP and then NH3.     

The pH dependence of hydrogen exchange in proteins

The static accessibility modified discrete charge model for electrostatic interactions in proteins is extended to the prediction of the pH dependence of hydrogen exchange reactions. The exchange rate profiles of buried amide protons are shown to follow the calculated pH dependence of the electrostatic component of protein stability. Rate profiles are calculated for individual buried amide protons in ribonuclease S and bovine pancreatic trypsin inhibitor. The electrostatic free energy of stabilization of the protein and the energy required to bring the catalytic ion to an exchange site are expressed as an apparent, pH-dependent contribution to the activation energy. Changes in the electrostatic stabilization of the proteins affect the calculated exchange rate for buried amide protons by more than 1000, while local field effects raise or lower the predicted exchange rates by less than 100. The pH dependence of exchangeable protons at the protein surface, such as the C-2 imidazole protons, is shown to follow the estimated energy required to introduce the catalytic ion at the exchange site. These calculations are discussed in terms of current models for proton exchange which incorporate the dynamic nature of the structure to explain exchange data from the interior of a protein.     

Structural basis for accelerated cleavage of bovine pancreatic trypsin inhibitor (BPTI) by human mesotrypsin

Human mesotrypsin is an isoform of trypsin that displays unusual resistance to polypeptide trypsin inhibitors and has been observed to cleave several such inhibitors as substrates. Whereas substitution of arginine for the highly conserved glycine 193 in the trypsin active site has been implicated as a critical factor in the inhibitor resistance of mesotrypsin, how this substitution leads to accelerated inhibitor cleavage is not clear. Bovine pancreatic trypsin inhibitor (BPTI) forms an extremely stable and cleavage-resistant complex with trypsin, and thus provides a rigorous challenge of mesotrypsin catalytic activity toward polypeptide inhibitors. Here, we report kinetic constants for mesotrypsin and the highly homologous (but inhibitor sensitive) human cationic trypsin, describing inhibition by, and cleavage of BPTI, as well as crystal structures of the mesotrypsin-BPTI and human cationic trypsin-BPTI complexes. We find that mesotrypsin cleaves BPTI with a rate constant accelerated 350-fold over that of human cationic trypsin and 150,000-fold over that of bovine trypsin. From the crystal structures, we see that small conformational adjustments limited to several side chains enable mesotrypsin-BPTI complex formation, surmounting the predicted steric clash introduced by Arg-193. Our results show that the mesotrypsin-BPTI interface favors catalysis through (a) electrostatic repulsion between the closely spaced mesotrypsin Arg-193 and BPTI Arg-17, and (b) elimination of two hydrogen bonds between the enzyme and the amine leaving group portion of BPTI. Our model predicts that these deleterious interactions accelerate leaving group dissociation and deacylation.     

Hydrogen exchange kinetics of surface peptide amides in bovine pancreatic trypsin inhibitor

The acid and base catalytic rate constants, kH, obs and kOH, obs and the pH at the minimum rate, pHmin, of 25 rapidly exchanging protons in bovine pancreatic trypsin inhibitor have been determined. Here we report the labeling procedure giving 1H nuclear magnetic resonance spectral resolution of seven additional rapidly exchanging NH protons and the pH dependence of their chemical shifts. Values of kH,obs kOH,obs and pHmin are given for Ala16, Gly28 and Arg53 NH groups, the only backbone amide protons with static accessibility of more than zero in the crystal structure not previously reports, and for Gly56 NH, buried at the C terminus of an alpha-helix. All four protons reported here have pH min greater than or equal to 3. Conclusions of the previous study predict that peptide protons with pHmin higher than those of model compounds have greater static accessibility of the peptide O than of the peptide N atom. The locations in the crystal structure of the four NH groups whose exchange rates are reported here are in qualitative agreement with these predictions. The ionic strength dependence of Ala16 at pH 5.5 shows a sharp increase in the exchange rate with decreasing salt concentration, as expected for base-catalyzed exchange in a positive electrostatic field.     

Substitutions at the P(1) position in BPTI strongly affect the association energy with serine proteinases

The role of the S(1) subsite in trypsin, chymotrypsin and plasmin has been examined by measuring the association with seven different mutants of bovine pancreatic trypsin inhibitor (BPTI); the mutants contain Gly, Ala, Ser, Val, Leu, Arg, and Trp at the P(1) position of the reactive site. The effects of substitutions at the P(1) position on the association constants are very large, comprising seven orders of magnitude for trypsin and plasmin, and over five orders for chymotrypsin. All mutants showed a decrease of the association constant to the three proteinases in the same order: Ala>Gly>Ser>Arg>Val>Leu>Trp. Calorimetric and circular dichroism methods showed that none of the P1 substitutions, except the P1-Val mutant, lead to destabilisation of the binding loop conformation. The X-ray structure of the complex formed between bovine beta-trypsin and P(1)-Leu BPTI showed that the P(1)-Leu sterically conflicts with the side-chain of P(3)-Ile, which thereby is forced to rotate approximately 90 degrees. Ile18 (P(3)) in its new orientation, in turn interacts with the Tyr39 side-chain of trypsin. Introduction of a large side-chain at the P1' position apparently leads to a cascade of small alterations of the trypsin-BPTI interface that seem to destabilise the complex by it adopting a less optimized packing and by tilting the BPTI molecule up to 15 degrees compared to the native trypsin-BPTI complex.     

The mechanism of glycogen synthetase as determined by deuterium isotope effects and positional isotope exchange experiments

The reaction mechanism for glycogen synthetase from rabbit muscle was examined by alpha-secondary deuterium isotope effects and positional exchange experiments. Incubation of glycogen synthetase with [beta-18O2,alpha beta-18O]UDP-Glc did not result in any detectable positional isotope exchange from the beta-nonbridge position to the anomeric oxygen of the glucose moiety. Glucono-1,5-lactone was found to be a noncompetitive inhibitor versus UDP-Glc. The kinetic constants, K(is) and K(ii), were found to be 91 +/- 4 microM and 0.70 +/- 0.09 mM, respectively. Deoxynojirimycin was a nonlinear inhibitor at pH 7.5. The alpha-secondary deuterium isotope effects were measured with [1-2H]UDP-Glc by the direct comparison method. The isotope effects on Vmax and Vmax/K were found to be 1.23 +/- 0.04 and 1.09 +/- 0.06, respectively. The inhibitory effects by glucono-lactone and deoxynojirimycon plus the large alpha-secondary isotope effect on Vmax have been interpreted to show that an oxocarbonium ion is an intermediate in this reaction mechanism. The lack of a detectable positional isotope exchange reaction in the absence of glycogen suggests the formation of a rigid tight ion pair between UDP and the oxocarbonium ion intermediate.     

Hydrogen exchange of lysozyme powders. Hydration dependence of internal motions

The rate of exchange of the labile hydrogens of lysozyme was measured by out-exchange of tritium from the protein in solution and from powder samples of varied hydration level, for pH 2, 3, 5, 7, and 10 at 25 degrees C. The dependence of exchange of powder samples on the level of hydration was the same for all pHs. Exchange increased strongly with increased hydration until reaching a rate of exchange that is constant above 0.15 g of H2O/g of protein (120 mol of H2O/mol of protein). This hydration level corresponds to coverage of less than half the protein surface with a monolayer of water. No additional hydrogen exchange was observed for protein powders with higher water content. Considered in conjunction with other lysozyme hydration data [Rupley, J. A., Gratton, E., & Careri, G. (1983) Trends Biochem. Sci. (Pers. Ed.) 8, 18-22], this observation indicates that internal protein dynamics are not strongly coupled to surface properties. The use of powder samples offers control of water activity through regulation of water vapor pressure. The dependence of the exchange rate on water activity was about fourth order. The order was pH independent and was constant from 114 to 8 mol of hydrogen remaining unexchanged/mol of lysozyme. These results indicate that the rate-determining step for protein hydrogen exchange is similar for all backbone amides and involves few water molecules. Powder samples were hydrated either by isopiestic equilibration, with a half-time for hydration of about 1 h, or by addition of solvent to rapidly reach final hydration. Samples hydrated slowly by isopiestic equilibration exhibited more exchange than was observed for samples of the same water content that had been hydrated rapidly by solvent addition. This difference can be explained by salt and pH effects on the nearly dry protein. Such effects would be expected to contribute more strongly during the isopiestic equilibration process. Solution hydrogen exchange measurements made for comparison with the powder measurements are in good agreement with published data. Rank order was proven the same for all pHs by solution pH jump experiments. The effect of ionic strength on hydrogen exchange was examined at pH 2 and pH 5 for protein solutions containing up to 1.0 M added salt. The influence of ionic strength was similar for both pHs and was complex in that the rate increased, but not monotonically, with increased ionic strength.     

NMR spectroscopy of hydroxyl protons in aqueous solutions of peptides and proteins

Summary Hydroxyl groups of serine and threonine, and to some extent also tyrosine are usually located on or near the surface of proteins. NMR observations of the hydroxyl protons is therefore of interest to support investigations of the protein surface in solution, and knowledge of the hydroxyl NMR lines is indispensable as a reference for studies of protein hydration in solution. In this paper, solvent suppression schemes recently developed for observation of hydration water resonances were used to observe hydroxyl protons of serine, threonine and tyrosine in aqueous solutions of small model peptides and the protein basic pancreatic trypsin inhibitor (BPTI). The chemical shifts of the hydroxyl protons of serine and threonine were found to be between 5.4 and 6.2 ppm, with random-coil shifts at 4°C of 5.92 ppm and 5.88 ppm, respectively, and those of tyrosine between 9.6 and 10.1 ppm, with a random-coil shift of 9.78 ppm. Since these spectral regions are virtually free of other polypeptide¹H NMR signals, cross peaks with the hydroxyl protons are usually well separated even in homonuclear two-dimensional¹H NMR spectra. To illustrate the practical use of hydroxyl proton NMR in polypeptides, the conformations of the side-chain hydroxyl groups in BPTI were characterized by measurements of nuclear Overhauser effects and scalar coupling constants involving the hydroxyl protons. In addition, hydroxyl proton exchange rates were measured as a function of pH, where simple first-order rate processes were observed for both acid- and base-catalysed exchange of all but one of the hydroxyl-bearing residues in BPTI. For the conformations of the individual Ser, Thr and Tyr side chains characterized in the solution structure with the use of hydroxyl proton NMR, both exact coincidence and significant differences relative to the corresponding BPTI crystal structure data were observed.[/p]     

Structure and dynamics of the neutrophil defensins NP-2, NP-5, and HNP-1: NMR studies of amide hydrogen exchange kinetics

The exchange kinetics for the slowly exchanging amide hydrogens in three defensins, rabbit NP-2, rabbit NP-5, and human HNP-1, have been measured over a range of pH at 25°C using 1D and 2D NMR methods. These NHs have exchange rates 10² to 10⁵ times slower than rates from unstructured model peptides. The observed distribution of exchange rates under these conditions can be rationalized by intramolecular hydrogen bonding of the individual NHs, solvent accessibility of the NHs, and local fluctuations in structure. The temperature dependencies of NH chemical shifts (NH temperature coefficients) were measured for the defensins and these values are consistent with the defensin structure. A comparison is made between NH exchange kinetics, NH solvent accessibility, and NH temperature coefficients of the defensins and other globular proteins. Titration of the histidine side chain in NP-2 was examined and the results are mapped to the three-dimensional structure. © 1994 Wiley-Liss, Inc.     

Kinetics of unfolding and folding from amide hydrogen exchange in native ubiquitin

Amide hydrogen (NH) exchange is one of the few experimental techniques with the potential for determining the thermodynamics and kinetics of conformational motions at nearly every residue in native proteins. Quantitative interpretation of NH exchange in terms of molecular motions relies on a simple two-state kinetic model: at any given slowly exchanging NH, a closed or exchange-incompetent conformation is in equilibrium with an open or exchange-competent conformation. Previous studies have demonstrated the accuracy of this model in measuring conformational equilibria by comparing exchange data with the thermodynamics of protein unfolding. We report here a test of the accuracy of the model in determining the kinetics of conformational changes in native proteins. The kinetics of folding and unfolding for ubiquitin have been measured by conventional methods and compared with those derived from a comprehensive analysis of the pH dependence of exchange in native ubiquitin. Rate constants for folding and unfolding from these two very different types of experiments show good agreement. The simple model for NH exchange thus appears to be a robust framework for obtaining quantitative information about molecular motions in native proteins.     

Raman dynamic probe of hydrogen exchange in bean pod mottle virus: base-specific retardation of exchange in packaged ssRNA.

We describe a novel approach to investigating exchange kinetics in biological assemblies. The method makes use of a Raman multichannel analyzer coupled with a dialysis flow cell. We employ this methodology to determine exchange rates of labile hydrogens in both the packaged RNA genome and protein subunits of bean pod mottle virus (BPMV). In the BPMV assembly, which is similar to human picornaviruses, the x-ray structure indicates that about 20% of the ssRNA chain is ordered at the threefold vertices of the icosahedral capsid, although the nucleotide bases in the ordered segments are not known (Chen et al., 1989). Here, we compare exchange profiles of the native virus with those of the empty capsid, model nucleic acids and aqueous solvent to reveal the following exchange characteristics of BPMV RNA and protein: (i) Base-specific retardation of exchange is observed in the packaged RNA. (ii) Retardation is greatest for uracil residues, for which the first-order exchange rate constant (kU = 0.18 +/- 0.02 min-1) is 40% lower than that of either the H2O solvent or adenine or cytosine groups of RNA (ksolv approximately kA approximately kC = 0.30 +/- 0.02 min-1). (iii) Retardation of exchange is also observed for the guanine residues of packaged RNA. (iv) No appreciable exchange of amide NH groups of capsid subunits occurs within the time of complete exchange (t approximately 10 min) of packaged RNA or bulk solvent. Thus, the present results identify sites in both the protein subunits (amide NH) and RNA nucleotides (amino NH2 and imino NH) which are resistant to solvent-catalyzed hydrogen exchange. We propose that retardation of exchange of labile sites of the RNA nucleotides is a consequence of the organization of the RNA chromosome within the virion. Our findings support a model for BPMV in which surface and buried domains of capsid subunits are extensively and rigidly hydrogen-bonded, and in which uracil and guanine exocyclic donor groups of packaged RNA are the principal targets for subunit interaction at the threefold vertices of the capsid.     

Synthesis and characterization of a pancreatic trypsin inhibitor homologue and a model inhibitor.

The synthesis and characterization of protein proteinase inhibitor homologues with variations in the amino acid composition in the vicinity of the reactive site should aid the understanding of the mechanism by which inhibition of enzymatic activity occurs. A homologue inhibitor in which the reactive-site residue Ala-16 of basic pancreatic trypsin inhibitor (Kunitz) (BPTI) is replaced by Phe has been synthesized to study the effect of this replacement on the dissociation constants of the enzyme-inhibitor complexes. The replacement of Ala-16 by Phe causes a dramatic increase in the K1 value of the trypsin-BPTI complex while that of the chymotrypsin-BPTI complex remains essentially the same. This cannot be explained simply in terms of increased steric crowding. The Phe replacement probably causes a small change in the local conformation of the reactive site of the inhibitor which leads to a large decrease in the stability of the very tight trypsin-BPTI complex. This conformation change apparently can be tolerated in the less tightly bound chymotrypsin-BPTI complex. On the basis of the known structure of BPTI, a cyclic heptadecapeptide containing one disulfide bond was synthesized as a model inhibitor in order to determine if a smaller peptide can be designed to act as a highly efficient inhibitor for trypsin. This heptadecapeptide which contains all of the amino acid residues of BPTI taking part in the interaction of the proteinase inhibitor with trypsin binds 3 X 10(7) time more weakly to the enzyme than native BPTI does. It thus appears that even though only a small part of the inhibitor molecule enters directly into interaction with the enzyme, the remaining portions of the molecule which hold the structure of the inhibitor rigid are essential for the strong interaction.