Structural characterization by nuclear magnetic resonance of a reactive-site 13carbon-labelled basic pancreatic trypsin inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed

With the use of an enzymatic replacement method, 90%-enriched [1-13C]lysine was introduced into the reactive site of the basic pancreatic trypsin inhibitor. Characterization of the labelled inhibitor with 13C nuclear magnetic resonance (NMR), 1H NMR and chemical methods showed that while the reactive-site peptide bond Lys-15--Ala-16 was properly resynthesized, the polypeptide chain was cleaved at the peptide bond Arg-39--Ala-40 and Arg-39 was removed. Detailed 1H NMR studies showed further that, with the exception of the immediate environment of the modification site, the average spatial structure of the native inhibitor was preserved in the modified protein. Compared to the native inhibitor, the thermal stability of the globular conformation was found to be reduced, interior amide protons exchanged at a faster rate and the internal mobility of aromatic rings located outside the immediate environment of the cleaved peptide bond was essentially unchanged. These observations coincide closely with previous reports on different modifications of the inhibitor and can be explained by a recently proposed dynamic multi-state model for globular proteins. Since the fundamental structural properties of the native inhibitor and full inhibitory activity are preserved after resynthesis, the [1-13C]lys-15-labelled inhibitor with the peptide bond Arg-39--Ala-40 cleaved and Arg-39 removed should be suitable for 13C NMR studies of mechanistic aspects of proteinase-inhibitor interactions.     

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.     

Binding of the Ile-Val and Val-Val effector dipeptides to the binary adducts of bovine trypsinogen with Kunitz and Kazal inhibitors as well as the acylating agent p-nitrophenyl p-guanidinobenzoate. A thermodynamic and kinetic study

Thermodynamics and kinetics of binding of the Ile-Val and Val-Val effector dipeptides to the binary adducts of bovine trypsinogen with the bovine basic pancreatic trypsin inhibitor (BPTI, Kunitz inhibitor), the porcine pancreatic secretory inhibitor (PSTI, Kazal inhibitor) and the acylating agent p-nitrophenyl p-guanidinobenzoate have been investigated at pH 7.4 and 21(+/- 0.5) degrees C. The affinity of both effector dipeptides for bovine trypsinogen: BPTI and bovine trypsinogen: PSTI binary adducts is higher than that observed for the formation of the dipeptide: bovine trypsinogen: p-guanidinobenzoate ternary complexes; moreover, the affinity of Ile-Val for the zymogen binary adducts is higher than that observed for Val-Val association. Binding of Ile-Val and Val-Val to the bovine trypsinogen binary complexes conforms to the induced-fit model, which consists of a fast pre-equilibrium followed by intramolecular isomerization change(s), the latter fast pre-equilibrium followed by intramolecular isomerization change(s), the latter representing the rate-limiting first-order process. For the three bovine trypsinogen systems considered, the rate of the intramolecular isomerization change(s) is essentially independent of the nature of the dipeptide and of the proenzyme binary complex.     

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.     

Kinetics of the interaction of alpha-chymotrypsin with trypsin kallikrein inhibitor (Kunitz) in which the reactive-site peptide bond Lys-15--Ala-16 is split.

Modified trypsin kallikrein inhibitor (I*), with the reactive-site peptide bond Lys-15--Ala-16 split, reacts with alpha-chymotrypsin (E) via an intermediate X to the stable tetrahedral complex C:E + I in equilibrium X leads to C. Formation X constitutes a fast pre-equilibrium (equilibrium constant Kx = 7 X 10(-5) M, association rate constant kx = 4 X 10(3)M-1s-1) to the slow reaction X leads to C (rate constant kc = 2 X 10(-3) s-1), all values at pH 7.5. No intermediate X is observed when alpha-chymotrypsin reacts with I*-OMe in which the carboxyl group of Lys-15 is esterified by methanol. This observation as well as the different pH dependence of the overall association rate constants in the case of I* and I*-OMe indicate tha formation of X precedes formation of the acyl enzyme in the catalytic pathway. The data are compared to the similar results obtained with beta-trypsin and I* or I*-OMe.     

Methods for dual, site-specific derivatization of bovine pancreatic trypsin inhibitor: trypsin protection of lysine-15 and attachment of fatty acids or hydrophobic peptides at the N-terminus

To produce a series of model membrane proteins, bovine pancreatic trypsin inhibitor (BPTI) has been modified by specifically attaching reporter groups to Lys-15 and fatty acids or hydrophobic peptides at the N-terminus. Lys-15 of BPTI was protected by trypsin bound to BPTI, then O-methylisourea (OMIU) was used to guanidinate all unprotected lysines. The N-terminal amine was then reacted with several saturated fatty acid anhydrides from 8 to 18 carbons in length, or with an SMCC cross-linker. Cysteine-containing hydrophobic peptides, cleaved from resin in the presence of sodium dodecyl sulfate (SDS), were then attached to the protein via the N-terminal cross-linker. The methods described yield a unique, chemically modified protein which can carry site-specific modifications at two distinct residues. The resulting proteins are ideal for diffusional or partitioning studies on model and biological membranes.     

Structure of the complex formed by bovine trypsin and bovine pancreatic trypsin inhibitor. Crystal structure determination and stereochemistry of the contact region

The structure of the complex of bovine trypsin and bovine pancreatic trypsin inhibitor has been determined by crystal structure analysis at 2.8 Å resolution. The structure is closely similar to the model predicted from the structures of the components. The complex is a tetrahedral adduct with a covalent bond between the carbonyl carbon of Lys-15I of the inhibitor and the γ-oxygen of Ser-195 of the enzyme. The imidazole of His-57 is hydrogen-bonded to Asp-102 and the bound seryl γ-oxygen in accord with the histidine being charged. The negatively charged carbonyl oxygen of Lys-15I forms two hydrogen bonds with the amide nitrogens of Gly-193 and Ser-195. Protonation of the leaving group N-H of Ala-16I to form an acyl-complex requires a conformational change of the imidazole of His-57. The tetrahedral adduct is further stabilized by hydrogen bonds between groups at the leaving group side and inhibitor and enzyme, which would be weakened in the acyl-enzyme. The kinetic data of inhibitor-enzyme interaction are reconciled with the structural model, and relations between enzyme-inhibitor interaction and productive enzyme-substrate interaction are proposed.     

Effect of Urey-Bradley-Shimanouchi force field on the harmonic dynamics of proteins.

A normal mode analysis of bovine pancreatic trypsin inhibitor is carried out by using a Urey-Bradley-Shimanouchi potential energy function. The density of vibrational states, the magnitudes, and time scales of the atomic fluctuations are compared with experimental and theoretical results obtained by the more commonly used potential energy functions. The atomic fluctuations of Lys-15 are subject to extensive considerations as this residue is buried in the trypsin specificity pocket. It is found that Arg-17 is likely to be of importance in order to understand the way BPTI binds on trypsin.     

Mesotrypsin Has Evolved Four Unique Residues to Cleave Trypsin Inhibitors as Substrates

Human mesotrypsin is highly homologous to other mammalian trypsins, and yet it is functionally unique in possessing resistance to inhibition by canonical serine protease inhibitors and in cleaving these inhibitors as preferred substrates. Arg-193 and Ser-39 have been identified as contributors to the inhibitor resistance and cleavage capability of mesotrypsin, but it is not known whether these residues fully account for the unusual properties of mesotrypsin. Here, we use human cationic trypsin as a template for engineering a gain of catalytic function, assessing mutants containing mesotrypsin-like mutations for resistance to inhibition by bovine pancreatic trypsin inhibitor (BPTI) and amyloid precursor protein Kunitz protease inhibitor (APPI), and for the ability to hydrolyze these inhibitors as substrates. We find that Arg-193 and Ser-39 are sufficient to confer mesotrypsin-like resistance to inhibition; however, compared with mesotrypsin, the trypsin-Y39S/G193R double mutant remains 10-fold slower at hydrolyzing BPTI and 2.5-fold slower at hydrolyzing APPI. We identify two additional residues in mesotrypsin, Lys-74 and Asp-97, which in concert with Arg-193 and Ser-39 confer the full catalytic capability of mesotrypsin for proteolysis of BPTI and APPI. Novel crystal structures of trypsin mutants in complex with BPTI suggest that these four residues function cooperatively to favor conformational dynamics that assist in dissociation of cleaved inhibitors. Our results reveal that efficient inhibitor cleavage is a complex capability to which at least four spatially separated residues of mesotrypsin contribute. These findings suggest that inhibitor cleavage represents a functional adaptation of mesotrypsin that may have evolved in response to positive selection pressure.     

High-level bacterial expression and 15N-alanine-labeling of bovine trypsin. Application to the study of trypsin-inhibitor complexes and trypsinogen activation by NMR spectroscopy

We describe here the high-level expression of bovine trypsinogen in E. coli, its refolding and activation to beta-trypsin, and the selective incorporation of (15)N-labeled alanine through supplementation of the growth medium. Using this procedure, we expressed (15)N-labeled S195A trypsinogens, both on a wild-type and on a D189S background, in amounts suitable for NMR spectroscopy. 2D [(1)H-(15)N]-HSQC NMR was used to follow conformational changes upon activation of trypsinogen and formation of noncovalent complexes between S195A or S195A/D189S trypsin and protein proteinase inhibitors of different structural families and different sizes, as well as to examine the effects of introduction of the D189S mutation. Spectra of good quality were obtained for both trypsins alone and in complexes of increasing size with the proteinase inhibitors BPTI (total molecular mass 31 kDa), SBTI (total molecular mass 44 kDa), and the serpin alpha(1)-proteinase inhibitor Pittsburgh (alpha(1)PI Pittsburgh) (total molecular mass 69 kDa). Assignments of alanines 55 and 56, close to the active site histidine, and of alanine 195, present in the S195A variant used for most of the studies, were made by mutagenesis. These three alanines, together with two others, probably close to the S1 specificity pocket, were very sensitive to complex formation. In contrast, the remaining 10 alanines were invariant in chemical shift in all 3 of the noncovalent complexes formed, reflecting the conservation of structure in complexes with BPTI and SBTI known from X-ray crystal structures, but also indicating that there is no change in backbone conformation for the noncovalent complex with alpha(1)PI, for which there is no crystal structure. This was true both for S195A and for S195A/D189S trypsins. This high-level expression and labeling approach will be of great use for solution NMR studies on trypsin-serpin complexes, as well as for structural and mechanistic studies on trypsin variants.     

Crystal structure of Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 in complex with trypsin. Implications for KD1 specificity of inhibition

Kunitz domain 1 (KD1) of tissue factor pathway inhibitor-2 inhibits trypsin, plasmin, and factor VIIa (FVIIa)/tissue factor with Ki values of 13, 3, and 1640 nM, respectively. To investigate the molecular specificity of KD1, crystals of the complex of KD1 with bovine beta-trypsin were obtained that diffracted to 1.8 A. The P1 residue Arg-15 (bovine pancreatic trypsin inhibitor numbering) in KD1 interacts with Asp-189 (chymotrypsin numbering) and with the carbonyl oxygens of Gly-219 and Ogamma of Ser-190. Leu-17, Leu-18, Leu-19, and Leu-34 in KD1 make van der Waals contacts with Tyr-39, Phe-41, and Tyr-151 in trypsin, forming a hydrophobic interface. Molecular modeling indicates that this complementary hydrophobic patch is composed of Phe-37, Met-39, and Phe-41 in plasmin, whereas in FVIIa/tissue factor, it is essentially absent. Arg-20, Tyr-46, and Glu-39 in KD1 interact with trypsin through ordered water molecules. In contrast, insertions in the 60-loop in plasmin and FVIIa allow Arg-20 of KD1 to directly interact with Glu-60 in plasmin and Asp-60 in FVIIa. Moreover, Tyr-46 in KD1 electrostatically interacts with Lys-60A and Arg-60D in plasmin and Lys-60A in FVIIa. Glu-39 in KD1 interacts directly with Arg-175 of the basic patch in plasmin, whereas in FVIIa, such interactions are not possible. Thus, the specificity of KD1 for plasmin is attributable to hydrophobic and direct electrostatic interactions. For trypsin, hydrophobic interactions are intact, and electrostatic interactions are weak, whereas for FVIIa, hydrophobic interactions are missing, and electrostatic interactions are partially intact. These findings provide insight into the protease selectivity of KD1.     

The effect of cleaving the reactive-site peptide bond Lys-15--Ala-16 on the conformation of bovine trypsin-kallikrein inhibitor (K unitz) as revealed by solvent-perturbation spectra, circular dichroism and fluorescence.

Spectroscopic measurements of virgin bovine trypsin-kallikrein inhibitor and its modified species (in which the reactive-site peptide bond Lys-15--Ala-16 is split) indicate a conformational difference between both proteins. The inhibitor contains four tyrosines but no tryptophan. In the modified inhibitor a tyrosyl blue shift is seen in the difference absorption spectrum of modified against virgin inhibitor. The solvent perturbation spectra show an increase of the fraction of exposed tyrosyls from 0.45 in the virgin inhibitor to 0.59 in the modified form. Comparison of the circular dichroism spectra of the modified and virgin inhibitors reveals a decrease of the mean residue ellipticity in the tyrosine and peptide bond region of the modified inhibitor. In the fluorescence spectra a 50% increase in the quantum yield of the tyrosine fluorescence is observed in the modified inhibitor. All these spectroscopic data support the idea, which is also evidenced by the X-ray crystallographic model, that in the modified inhibitor up to five residues from Ala-16 to Arg-20 gain rotational freedom.     

Engineering trypsin for inhibitor resistance

The development of effective protease therapeutics requires that the proteases be more resistant to naturally occurring inhibitors while maintaining catalytic activity. A key step in developing inhibitor resistance is the identification of key residues in protease-inhibitor interaction. Given that majority of the protease therapeutics currently in use are trypsin-fold, trypsin itself serves as an ideal model for studying protease-inhibitor interaction. To test the importance of several trypsin-inhibitor interactions on the prime-side binding interface, we created four trypsin single variants Y39A, Y39F, K60A, and K60V and report biochemical sensitivity against bovine pancreatic trypsin inhibitor (BPTI) and M84R ecotin. All variants retained catalytic activity against small, commercially available peptide substrates [k_cat/K_M = (1.2 ± 0.3) × 10⁷ M⁻¹ s⁻¹. Compared with wild-type, the K60A and K60V variants showed increased sensitivity to BPTI but less sensitivity to ecotin. The Y39A variant was less sensitive to BPTI and ecotin while the Y39F variant was more sensitive to both. The relative binding free energies between BPTI complexes with WT, Y39F, and Y39A were calculated based on 3.5 µs combined explicit solvent molecular dynamics simulations. The BPTI:Y39F complex resulted in the lowest binding energy, while BPTI:Y39A resulted in the highest. Simulations of Y39F revealed increased conformational rearrangement of F39, which allowed formation of a new hydrogen bond between BPTI R17 and H40 of the variant. All together, these data suggest that positions 39 and 60 are key for inhibitor binding to trypsin, and likely more trypsin-fold proteases.     

Site-directed mutagenesis of human pancreatic secretory trypsin inhibitor

Arg-42 or Lys-43 or Arg-44 of human pancreatic secretory trypsin inhibitor (PSTI) was replaced by Thr or Ser by site-directed mutagenesis, and the inactivation rates of the mutants after mixing with human trypsin were compared with that of the natural form. The inactivation rate decreased for one mutant (Arg-44----Ser), whereas no change was observed for another (Arg-42----Thr) and an increase was observed for a third (Lys-43----Thr). Kinetic studies on the interactions between human trypsin and synthetic peptides, comprising the regions of Phe39-Ser47 of the respective PSTI species, showed that human trypsin cleaved the Arg42-Lys43 bond preferentially to the Arg44-Gln45 bond. However, it is cleavage of the latter bond that is thought to cause inactivation of human PSTI. These results suggest that the Arg44-Gln45 bond of human PSTI is responsible for its inhibitory activity, and inactivation of human PSTI is probably caused by deletion of the dipeptide Lys43-Arg44.     

Production of native, correctly folded bovine pancreatic trypsin inhibitor by Escherichia coli

A gene for bovine pancreatic trypsin inhibitor (BPTI) was fused to the coding sequence for the Escherichia coli alkaline phosphatase signal peptide and expressed in E. coli under the control of the alkaline phosphatase promoter. When induced in phosphate-depleted medium such cells produced a trypsin inhibitor that was indistinguishable from native, properly folded BPTI. In particular, the BPTI produced by E. coli had three disulfide bonds that appeared to be identical to those found in native BPTI, as assayed by sensitivity to iodoacetate, dithiothreitol, and urea. This expression/secretion system will make possible the production of variant BPTI molecules, thus allowing the perturbing effects of amino acid substitutions on BPTI folding, structure, and function to be assessed.     

Comparison of anionic and cationic trypsinogens: the anionic activation domain is more flexible in solution and differs in its mode of BPTI binding in the crystal structure

Unlike bovine cationic trypsin, rat anionic trypsin retains activity at high pH. This alkaline stability has been attributed to stabilization of the salt bridge between the N-terminal Ile16 and Asp194 by the surface negative charge (Soman K, Yang A-S, Honig B, Fletterick R., 1989, Biochemistry 28:9918-9926). The formation of this salt bridge controls the conformation of the activation domain in trypsin. In this work we probe the structure of rat trypsinogen to determine the effects of the surface negative charge on the activation domain in the absence of the Ile16-Asp194 salt bridge. We determined the crystal structures of the rat trypsin-BPTI complex and the rat trypsinogen-BPTI complex at 1.8 and 2.2 A, respectively. The BPTI complex of rat trypsinogen resembles that of rat trypsin. Surprisingly, the side chain of Ile16 is found in a similar position in both the rat trypsin and trypsinogen complexes, although it is not the N-terminal residue and cannot form the salt bridge in trypsinogen. The resulting position of the activation peptide alters the conformation of the adjacent autolysis loop (residues 142-153). While bovine trypsinogen and trypsin have similar CD spectra, the CD spectrum of rat trypsinogen has only 60% of the intensity of rat trypsin. This lower intensity most likely results from increased flexibility around two conserved tryptophans, which are adjacent to the activation domain. The NMR spectrum of rat trypsinogen contains high field methyl signals as observed in bovine trypsinogen. It is concluded that the activation domain of rat trypsinogen is more flexible than that of bovine trypsinogen, but does not extend further into the protein core.     

Protease inhibitor display M13 phage: selection of high-affinity neutrophil elastase inhibitors.

We report display of the complete protease inhibitor (Kunitz) domain, BPTI, on the surface of bacteriophage M13 as a fusion to the gene III product. Phage that display BPTI bind specifically to anti-BPTI antibodies, trypsin and anhydrotrypsin. A point mutation of BPTI [Lys15-->Leu(K15L)] alters the binding specificity of fusion phage such that a human neutrophil elastase-binding phenotype is conferred while a trypsin-binding phenotype is eliminated. Phage were eluted from an immobilized protease with step gradients of decreasing pH. Phage that display Kunitz domains having higher affinity for the immobilized protease exhibit characteristic pH elution phenotypes, indicating that bound display phage can be selectively recovered from an affinity matrix. Utilization of this technology should enable the selection of remodeled protease inhibitors exhibiting novel binding specificities.     

Design, construction and function of a multicopy display vector using fusions to the major coat protein of bacteriophage M13.

Incorporation of numerous copies of a heterologous protein (bovine pancreatic trypsin inhibitor; BPTI) fused to the mature major coat protein (gene VIII product; VIII) of bacteriophage M13 has been demonstrated. Optimization of the promoter, signal peptide and host bacterial strain allowed for the construction of a working vector consisting of the M13 genome, into which was cloned a synthetic gene composed of a lac (or tac) promoter, and sequences encoding the bacterial alkaline phosphatase signal peptide, mature BPTI and the mature coat protein. Processing of the BPTI-VIII fusion protein and its incorporation into the bacteriophage were found to be maximal in a host bacterial strain containing a prlA/secY mutation. Functional protein is displayed on the surface of M13 phage, as judged by specific interactions with antiserum, anhydrotrypsin, and trypsin. Such display vectors can be used for epitope mapping, production of artificial vaccines and the screening of diverse libraries of proteins or peptides having affinity for a chosen ligand. The VIII display phage system has practical advantages over the III display phage system in that many more copies of the fusion protein can be displayed per phage particle and the presence of the VII fusion protein has little or no effect on the infectivity of the resulting bacteriophage.     

Rigidification of a flexible protease inhibitor variant upon binding to trypsin

The Tyr35-->Gly replacement in bovine pancreatic trypsin inhibitor (BPTI) has previously been shown to dramatically enhance the flexibility of the trypsin-binding region of the free inhibitor and to destabilize the interaction with the protease by about 3 kcal/mol. The effects of this replacement on the enzyme-inhibitor interaction were further studied here by X-ray crystallography and isothermal titration calorimetry (ITC). The co-crystal structure of Y35G BPTI bound to trypsin was determined using 1.65 A resolution X-ray diffraction data collected from cryopreserved crystals, and a new structure of the complex with wild-type BPTI under the same conditions was determined using 1.62 A data. These structures reveal that, in contrast to the free protein, Y35G BPTI adopts a conformation nearly identical with that of the wild-type protein, with a water-filled cavity in place of the missing Tyr side-chain. The crystallographic temperature factors for the two complexes indicate that the mutant inhibitor is nearly as rigid as the wild-type protein when bound to trypsin. Calorimetric measurements show that the change in enthalpy upon dissociation of the complex is 2.5 kcal/mol less favorable for the complex containing Y35G BPTI than for the complex with the wild-type inhibitor. Thus, the destabilization of the complex resulting from the Y35G replacement is due to a more favorable change in entropy upon dissociation. The heat capacity changes for dissociation of the mutant and wild-type complexes were very similar, suggesting that the entropic effects probably do not arise from solvation effects, but are more likely due to an increase in protein conformational entropy upon dissociation of the mutant inhibitor. These results define the biophysical role of a highly conserved core residue located outside of a protein-binding interface, demonstrating that Tyr35 has little impact on the trypsin-bound BPTI structure and acts primarily to define the structure of the free protein so as to maximize binding affinity.     

Evidence from 13C NMR for polarization of the carbonyl of oxaloacetate in the active site of citrate synthase

The carbon-13 NMR spectrum of oxaloacetate bound in the active site of citrate synthase has been obtained at 90.56 MHz. In the binary complex with enzyme, the positions of the resonances of oxaloacetate are shifted relative to those of the free ligand as follows: C-1 (carboxylate), -2.5 ppm; C-2 (carbonyl), +4.3 ppm; C-3 (methylene), -0.6 ppm; C-4 (carboxylate), +1.3 ppm. The change observed in the carbonyl chemical shift is successively increased in ternary complexes with the product [coenzyme A (CoA)], a substrate analogue (S-acetonyl-CoA), and an acetyl-CoA enolate analogue (carboxymethyl-CoA), reaching a value of +6.8 ppm from the free carbonyl resonance. Binary complexes are in intermediate to fast exchange on the NMR time scale with free oxaloacetate; ternary complexes are in slow exchange. Line widths of the methylene resonance in the ternary complexes suggest complete immobilization of oxaloacetate in the active site. Analysis of line widths in the binary complex suggests the existence of a dynamic equilibrium between two or more forms of bound oxaloacetate, primarily involving C-4. The changes in chemical shifts of the carbonyl carbon indicate strong polarization of the carbonyl bond or protonation of the carbonyl oxygen. Some of this carbonyl polarization occurs even in the binary complex. Development of positive charge on the carbonyl carbon enhances reactivity toward condensation with the carbanion/enolate of acetyl-CoA in the mechanism which has been postulated for this enzyme. The very large change in the chemical shift of the reacting carbonyl in the presence of an analogue of the enolate of acetyl-CoA supports this interpretation.