Formation of a trypsin-borate-4-aminobutanol ternary complex

The formation of ternary complexes involving serine proteases, borate, and an alcohol has important implications for understanding the physiological actions of borate and for the development of tight binding inhibitors for this class of enzymes. Recent studies of a related enzyme, gamma-glutamyl transpeptidase, which is subject to inhibition by a labile serine/borate mixture, have demonstrated that construction of a non-labile boronate analogue results in an inhibitor with nearly 10(5)-fold greater potency. To evaluate the generalization of this biochemistry to serine proteases, we have observed the ternary complex formed from 4-aminobutanol, borate, and trypsin. A combination of (11)B and (1)H NMR and spectrophotometric assays using acetylarginine p-nitroanilide (Ac-Arg-pNA) as the chromogenic substrate all indicate a cooperative binding interaction in which the borate is esterified by the oxygen atoms of the 4-aminobutanol and trypsin residue Ser(195). Two downfield-shifted proton resonances at 15.5 and 16.6 ppm are proposed to arise from the labile imidazolium protons on His(57), indicating a salt bridge interaction with the negatively charged borate. A cooperativity parameter alpha of 0.2 is derived from the assays. These results provide the first direct evidence for formation of a ternary complex involving a serine protease, borate, and an alcohol, and suggest that this represents a general approach for the development of tight binding ligands.     

NMR and crystallographic characterization of adventitious borate binding by trypsin

Recent 11B NMR studies of the formation of ternary complexes of trypsin, borate, and S1-binding alcohols revealed evidence for an additional binding interaction external to the enzyme active site. We have explored this binding interaction as a prototypical interaction of borate and boronate ligands with residues on the protein surface. NMR studies of trypsin in which the active site is blocked with leupeptin or with the irreversible inhibitor 4-(2-aminoethyl) benzenesulfonyl fluoride hydrochloride (AEBSF) indicate the existence of a low-affinity borate binding site with an apparent dissociation constant of 97 mM, measured at pH 8.0. Observation of a field-dependent dynamic frequency shift of the (11)B resonance indicates that it corresponds to a complex for which omegatau > 1. The 0.12 ppm shift difference of the borate resonances measured at 11.75 and 7.05 T, corresponds to a quadrupole coupling constant of 260 kHz. A much larger 2.0 ppm shift is observed in the 11B NMR spectra of trypsin complexed with benzene boronic acid (BBA), leading to a calculated quadrupole coupling constant of 1.1 MHz for this complex. Crystallographic studies identify the second borate binding site as a serine-rich region on the surface of the molecule. Specifically, a complex obtained at pH 10.6 shows a borate ion covalently bonded to the hydroxyl oxygen atoms of Ser164 and Ser167, with additional stabilization coming from two hydrogen-bonding interactions. A similar structure, although with low occupancy (30%), is observed for a trypsin-BBA complex. In this case, the BBA is also observed in the active site, covalently bound in two different conformations to both His57 Nepsilon and Ser195 Ogamma. An analysis of pairwise hydroxyl oxygen distances was able to predict the secondary borate binding site in porcine trypsin, and this approach is potentially useful for prediction of borate binding sites on the surfaces of other proteins. However, the distances between the Ser164/Ser167 Ogamma atoms in all of the reported trypsin crystal structures is significantly greater than the Ogamma distances of 2.2 and 1.9 angstroms observed in the trypsin complexes with borate and BBA, respectively. Thus, the ability of the hydroxyl oxygens to adopt a sufficiently close orientation to allow bidentate ligation is a critical limit on the borate binding affinity of surface-accessible serine/threonine/tyrosine residues.     

The ternary complex of Pseudomonas aeruginosa alcohol dehydrogenase with NADH and ethylene glycol

Pseudomonas aeruginosa alcohol dehydrogenase (PaADH; ADH, EC 1.1.1.1) catalyzes the reversible oxidation of primary and secondary alcohols to the corresponding aldehydes and ketones, using NAD as coenzyme. We crystallized the ternary complex of PaADH with its coenzyme and a substrate molecule and determined its structure at a resolution of 2.3 A, using the molecular replacement method. The PaADH tetramer comprises four identical chains of 342 amino acid residues each and obeys ~222-point symmetry. The PaADH monomer is structurally similar to alcohol dehydrogenase monomers from vertebrates, archaea, and bacteria. The stabilization of the ternary complex of PaADH, the coenzyme, and the poor substrate ethylene glycol (k(cat) = 4.5 sec(-1); Km > 200 mM) was due to the blocked exit of the coenzyme in the crystalline state, combined with a high (2.5 M) concentration of the substrate. The structure of the ternary complex presents the precise geometry of the Zn coordination complex, the proton-shuttling system, and the hydride transfer path. The ternary complex structure also suggests that the low efficiency of ethylene glycol as a substrate results from the presence of a second hydroxyl group in this molecule.     

Carbon-13 nuclear magnetic resonance studies of the selectively isotope-labeled reactive site peptide bond of the basic pancreatic trypsin inhibitor in the complexes with trypsin, trypsinogen, and anhydrotrypsin

A previously characterized modification of the basic pancreatic trypsin inhibitor (BPTI), with the carbonyl carbon atom of Lys-15 selectively enriched in 13C, the peptide bond Arg-39--Ala-40 cleaved, and Arg-39 removed, was used for 13C NMR studies of the reactive site peptide bond Lys-15--Ala-16 in the complexes with trypsin, trypsinogen, and anhydrotrypsin. The chemical shift of [1-13C]Lys-15 was 175.7 ppm in the free inhibitor, 176.4 ppm in the complexes with trypsin and anhydrotrypsin and the ternary complex with trypsinogen and H-Ile-Val-OH, and 175.7 ppm in a neutral solution containing the inhibitor and trypsinogen. These data show that the trypsin--BPTI complex does not contain a covalent tetrahedral carbon atom in the position of the reactive site peptide carbonyl of the inhibitor. They would be consistent with the formation of a noncovalent complex but cannot at present be used to further characterize the degree of a possible pyramidalization of the carbonyl carbon of Lys-15 in such a complex. The identical chemical shifts in the complexes with trypsin and anhydrotrypsin indicate that the gamma-hydroxyl group of Ser-195 of trypsin does not have an important role in the binding of the inhibitor. The previously described [Perkins, S. J. & Wüthrich, K. (1980) J. Mol. Biol. 138, 43--64] stepwise transition from the trypsinogen conformation to an intermediate conformational state in the trypsinogen--BPTI complex and a trypsin-like conformation in the ternary complex trypsinogen--BPTI--H-Ile-Val-OH appears to be manifested also in the chemical shift of [1-13C]Lys-15 of labeled BPTI.     

PEG and mPEG-anthracene conjugate with trypsin and trypsin inhibitor: hydrophobic and hydrophilic contacts

The conjugation of trypsin (try) and trypsin inhibitor (tryi) with poly(ethylene glycol) (PEG) and methoxypoly(ethylene glycol) anthracene (mPEG-anthracene) was investigated in aqueous solution, using multiple spectroscopic methods, thermodynamic analysis, and molecular modeling. Thermodynamic parameters ΔS, ΔH, and ΔG showed protein-PEG bindings occur via H-bonding and van der Waals contacts with trypsin inhibitor forming more stable conjugate than trypsin. As polymer size increased more stable PEG-protein conjugate formed, while hydrophobic mPEG-anthracene forms less stable protein complexes. Modeling showed the presence of several H-bonding contacts between polymer and amino acids that stabilize protein-polymer conjugation. Polymer complexation induces more perturbations of trypsin inhibitor structure than trypsin with reduction of protein alpha-helix and major increase in random structures, indicating protein structural destabilization.     

Short heparin sequences spaced by glycol-split uronate residues are antagonists of fibroblast growth factor 2 and angiogenesis inhibitors

Fibroblast Growth Factor-2 (FGF2) is a major inducer of neovascularization (angiogenesis). Heparin activates FGF2 by favoring formation of ternary complexes with its cellular receptors (FGFRs). Controlled 2-O-desulfation followed by exhaustive periodate oxidation/borohydride reduction has been used to generate sulfation gaps within the prevalent heparin sequences, building-up arrays of pentasulfated trisaccharides (PST, consisting of a 2-O-sulfated iduronic acid flanked by two N,6-disulfated glucosamines) spaced by reduced, glycol-split uronic acid (sU) residues. The structure of the prevalent sequences of the novel heparin derivative has been confirmed by mono- and two-dimensional NMR analysis. NMR spin-lattice relaxation times (T2) and nuclear Overhauser effects suggest that the sU residues act as flexible joints between the PST sequences and cause a marked distortion of the chain conformation of heparin required for formation of ternary complexes. Since the splitting reaction also occurs at the level of the essential glucuronic acid residue of the active site for antithrombin, the heparin derivative has no anticoagulant activity. However, it fully retains the FGF2-binding ability of the original heparin, as shown by its capacity to protect FGF2 from trypsin cleavage and to prevent the formation of heparan sulfate proteoglycan (HSPG)/FGF2/FGFR1 ternary complexes. However, when compared to heparin it showed a reduced capacity to induce FGF2 dimerization and to favor the interaction of [125I]FGF2 with FGFR1 in HSPG-deficient, FGFR1-transfected CHO cells. Accordingly, it was more effective than heparin in inhibiting the mitogenic activity exerted by FGF2 in cultured endothelial cells. Finally, it inhibited angiogenesis in a chick embrio chorioallantoic membrane (CAM) assay in which heparin is inactive.     

Formation of a noncovalent serpin-proteinase complex involves no conformational change in the serpin. Use of 1H-15N HSQC NMR as a sensitive nonperturbing monitor of conformation

A structural understanding of the nature and scope of serpin inhibition mechanisms has been limited by the inability so far to crystallize any serpin-proteinase complex. We describe here the application of [(1)H-(15)N]-HSQC NMR on uniformly and residue-selectively (15)N-labeled serpin alpha(1)-proteinase inhibitor (Pittsburgh variant with stabilizing mutations) to provide a nonperturbing and exquisitely sensitive means of probing the conformation of the serpin alone and in a noncovalent complex with inactive, serine 195-modified, bovine trypsin. The latter should be a good model both for the few examples of reversible serpin-proteinase complexes and for the initial Michaelis-like complex formed en route to irreversible covalent inhibition. Cleavage of the reactive center loop, with subsequent insertion into beta-sheet A, caused dramatic perturbation of most of the NMR cross-peaks. This was true for both the uniformly labeled and alanine-specifically labeled samples. The spectra of uniformly or leucine- or alanine-specifically labeled alpha(1)-proteinase inhibitor in noncovalent complex with unlabeled inactive trypsin gave almost no detectable chemical shift changes of cross-peaks, but some general increase in line width. Residue-specific assignments of the four alanines in the reactive center loop, at P12, P11, P9, and P4, allowed specific examination of the behavior of the reactive center loop. All four alanines showed higher mobility than the body of the serpin, consistent with a flexible reactive center loop, which remained flexible even in the noncovalent complex with proteinase. The three alanines near the hinge point for insertion showed almost no chemical shift perturbation upon noncovalent complex formation, while the alanine at P4 was perturbed, presumably by interaction with the active site of bound trypsin. Reporters from both the body of the serpin and the reactive center loop therefore indicate that noncovalent complex formation involves no conformational change in the body of the serpin and only minor perturbation of the reactive center loop in the region which contacts proteinase. Thus, despite the large size of serpin and serpin-proteinase complex, 45 and 69 kDa respectively, NMR provides a very sensitive means of probing serpin conformation and mobility, which should be applicable both to noncovalent and to covalent complexes with a range of different proteinases, and probably to other serpins.     

Interaction between dry starch and plasticisers glycerol or ethylene glycol, measured by differential scanning calorimetry and solid state NMR spectroscopy

The interaction of crystalline amylose and of crystalline and amorphous amylopectin with the plasticisers glycerol or ethylene glycol in the absence of water was studied, by using differential scanning calorimetry (DSC) and solid state nuclear magnetic resonance (NMR) spectroscopy. Upon heating starch freshly mixed with plasticisers, a strong exothermal interaction enthalpy of ΔH−35 J/g was detected by DSC. At room temperature glycerol interacts mainly with the amorphous starch regions, the interaction taking 8 days to reach equilibrium. For ethylene glycol the interaction is faster, taking four days to reach equilibrium, and the rate is not affected by crystallinity. Ethylene glycol interacts in a more ordered manner with amorphous than with crystalline material, resulting in a narrower ethylene glycol cross-polarisation magic angle spinning (CP/MAS) signal when equilibrium is reached at room temperature. Upon heating, more glycerol or ethylene glycol is immobilised, but in a less ordered manner than upon storage at room temperature. This results in a more intense, but broader plasticiser CP/MAS signal upon heating. Interaction in a more ordered manner probably implies interaction with more of the hydroxy groups of the plasticiser. The polysaccharide mobility is increased more when the plasticiser interacts in a more ordered manner, as observed by small starch signals in HP/DEC spectra.     

Supramolecular gene delivery vectors showing enhanced transgene expression and good biocompatibility

Soluble supramolecular inclusion complexes were formed by threading alpha-cyclodextrin (alpha-CD) molecules over poly(ethylene glycol) (PEG) and poly(epsilon-caprolactone) (PCL) chains of ternary block copolymers of PEG, PCL and polyethylenimine (PEI). Characteristic shifts of PCL absorptions in FTIR, (1)H NMR and UV spectra strongly suggest that alpha-CD is threaded over PEG and PCL blocks. Due to the reduced hydrophobic interaction between PCL blocks, the resulting supramolecular complexes displayed a dramatically increased solubility, in comparison with the ternary block copolymers. Their ability to complex DNA was almost as efficient as that of branched PEI 25 kDa, as shown in the ethidium bromide fluorescence quenching experiments. Resulting DNA polyplexes displayed a size of around 200 nm and a neutral surface charge. Microscopy studies in 3T3 fibroblasts revealed an efficient cellular uptake. Transfection efficiencies of inclusion complexes were in the same order of magnitude as PEI. In contrast to PEI a 100x lower toxicity was observed by MTT-assay, allowing the administration of nitrogen-to-phosphate ratios of up to 20. These new gene delivery systems merit further characterization under in vivo conditions.     

Modification of the interactions of myosin with actin and 5''-adenylyl imidodiphosphate by substitution of ethylene glycol for water.

We studied the effect of replacing water by ethylene glycol as solvent on the properties of skeletal muscle myosin, myosin subfragment-1 (S1) and heavy meromyosin. Ethylene glycol (50%, v/v) had no detectable effect on the affinity of myosin or actomyosin for the substrate analogue 5'-adenylyl imidodiphosphate (AMPPNP). However, the rate constants for formation and dissociation of the myosin X MgAMPPNP complex were reduced 200-fold; the logarithm of the dissociation rate was roughly proportional to the fractional concentration of ethylene glycol. Nucleotide dissociation was accelerated at least 300-fold by pure actin but remained slow with regulated actin in the absence of Ca2+. Ethylene glycol substitution reduced the affinity of S1 and the S1 X MgAMPPNP complex for actin equally (100-fold at 50% ethylene glycol). These results show that ethylene glycol has specific effects on myosin's enzymic mechanism, which can account for its effect on the tension and stiffness of glycerinated muscle fibres.     

Double-headed protease inhibitors from black-eyed peas. VI. Singlet-singlet energy transfer and other optical studies on the structure of trypsin and chymotrypsin complexes.

The geometry of the binary and ternary complexes of two black-eyed pea inhibitors with trypsin and chymotrypsin has been established by distance measurements using the technique of singlet-singlet energy transfer. Triangulation of measured distances in the ternary double-headed complex of the trypsin-chymotrypsin inhibitor (BEPCI) with trypsin and chymotrypsin limits the possible structural models for this complex to those in which the center to center distance between trypsin and chymotrypsin is about 64 A, the distance from the center of trypsin to the single fluorescently labeled tyrosyl residue of the BEPCI dimer is about 33 A, and the distance between the chymotrypsin center and the labeled tyrosine of the inhibitor is about 43 A. Energy transfer results for the trypsin inhibitor (BEPTI) complexes show conclusively that the weak trypsin site is structurally analogous to the strong chymotrypsin binding site of BEPCI. The weak chymotrypsin binding site of BEPTI is structurally analogous to the strong trypsin sites of BEPCI and BEPTI. Corresponding distances in binary and ternary complexes are the same, indicating that little or no structural rearrangement occurs when the ternary complexes are formed. Complex formation was shown to involve tryptophan and tryosine residues of both trypsin and chymotrypsin as judged by absorption and circular dichroism difference spectroscopy. In addition, circular dichroism difference spectra revealed some disulfide contributions.     

Crystal structure of the Bowman-Birk Inhibitor from Vigna unguiculata seeds in complex with beta-trypsin at 1.55 A resolution and its structural properties in association with proteinases

The structure of the Bowman-Birk inhibitor from Vigna unguiculata seeds (BTCI) in complex with β-trypsin was solved and refined at 1.55 Å to a crystallographic R_factor of 0.154 and R_free of 0.169, and represents the highest resolution for a Bowman-Birk inhibitor structure to date. The BTCI-trypsin interface is stabilized by hydrophobic contacts and hydrogen bonds, involving two waters and a polyethylene glycol molecule. The conformational rigidity of the reactive loop is characteristic of the specificity against trypsin, while hydrophobicity and conformational mobility of the antichymotryptic subdomain confer the self-association tendency, indicated by atomic force microscopy, of BTCI in complex and free form. When BTCI is in binary complexes, no significant differences in inhibition constants for producing a ternary complex with trypsin and chymotrypsin were detected. These results indicate that binary complexes present no conformational change in their reactive site for both enzymes confirming that these sites are structurally independent. The free chymotrypsin observed in the atomic force microscopy assays, when the ternary complex is obtained from BTCI-trypsin binary complex and chymotrypsin, could be related more to the self-association tendency between chymotrypsin molecules and the flexibility of the reactive site for this enzyme than to binding-related conformational changes.     

Ligand-induced conformational changes in the crystal structures of Pneumocystis carinii dihydrofolate reductase complexes with folate and NADP+

Structural data from two independent crystal forms (P212121 and P21) of the folate (FA) binary complex and from the ternary complex with the oxidized coenzyme, NADP+, and recombinant Pneumocystis carinii dihydrofolate reductase (pcDHFR) refined to an average of 2.15 A resolution, show the first evidence of ligand-induced conformational changes in the structure of pcDHFR. These data are also compared with the crystal structure of the ternary complex of methotrexate (MTX) with NADPH and pcDHFR in the monoclinic lattice with data to 2.5 A resolution. Comparison of the data for the FA binary complex of pcDHFR with those for the ternary structures reveals significant differences, with a >7 A movement of the loop region near residue 23 that results in a new "flap-open" position for the binary complex, and a "closed" position in the ternary complexes, similar to that reported for Escherichia coli (ec) DHFR complexes. In the orthorhombic lattice for the binary FA pcDHFR complex, there is also an unwinding of a short helical region near residue 47 that places hydrophobic residues Phe-46 and Phe-49 toward the outer surface, a conformation that is stabilized by intermolecular packing contacts. The pyrophosphate moiety of NADP+ in the ternary folate pcDHFR complexes shows significant differences in conformation compared with that observed in the MTX-NADPH-pcDHFR ternary complex. Additionally, comparison of the conformations among these four pcDHFR structures reveals evidence for subdomain movement that correlates with cofactor binding states. The larger binding site access in the new "flap-open" loop 23 conformation of the binary FA complex is consistent with the rapid release of cofactor from the product complex during catalysis as well as the more rapid release of substrate product from the binary complex as a result of the weaker contacts of the closed loop 23 conformation, compared to ecDHFR.     

Zinc-activated alcohols in ternary complexes of liver alcohol dehydrogenase

Activation parameters for each reaction step in the kinetic mechanism of liver alcohol dehydrogenase have been measured for the oxidation of ethanol and the reduction of acetaldehyde. In the oxidation process, the highest enthalpy of activation, 9.7 kcal/mol, occurs for the turnover of the liver alcohol dehydrogenase-NAD(+)-ethanol ternary complex. To investigate if this enthalpy requirement represents a change in the ionization state of ethanol bound in the ternary complex, inhibition of ethanol oxidation was determined using the following series of small, electronegative alcohols with pKa values ranging from 12.37 to 15.5: 2,2,2-trifluoroethanol, 2,2,2-trichloroethanol, 2,2,2-tribromoethanol, 2,2-dichloroethanol, 2,2-difluoroethanol, propargyl alcohol, 3-hydroxypropionitrile, 2-chloroethanol, 2-iodoethanol, 2-methoxyethanol, ethylene glycol, and methanol. The observed inhibition patterns were analyzed according to several kinetic inhibition models; in each case, the best fit model was used to determine the substrate competitive inhibition constant. A plot of the logarithm of these inhibition constants is shown to be dependent on the pKa values of the inhibiting alcohols with a slope approaching -1, indicating that inhibition is controlled by a proton loss from the alcohol. The observed competitive inhibition behavior, coupled with crystallographic studies depicting a direct ligation of an alcohol oxygen to the catalytic zinc ion, indicates that inhibition is controlled by the formation of a zinc-bound alkoxide. Because the inhibiting alcohols are structurally homologous to ethanol, a relationship between the inhibition constant and the inhibiting alcohol's pKa can be derived to show that the pKa of an alcohol bound in a ternary complex is also dependent on its pKa as a free alcohol. Ternary complex pKa values have been determined for ethanol and the inhibiting alcohols.     

Circular dichroism and circular polarization of luminescence of reduced nicotinamide adenine dinucleotide in solution and bound to dehydrogenases.

The CD (circular dichroism) and CPL (circular polarization of luminescence) spectra of NADPH in aqueous solution were studied and found to be markedly different. The spectra were not affected by cleavage of the coenzyme molecule with phosphodiesterase. The differences are thus not due to the existence of extended and folded conformations of NADPH and it is concluded that they originate in excited state conformational changes of the nicotinamide--ribose fragment. Opposite signs of both the CD and CPL spectra were observed for NADH bound to horse liver alcohol dehydrogenase and to beef heart lactate dehydrogenase indicating structural differences between the nicotinamide binding sites. The binding of substrate analogues to enzyme--coenzyme complexes did not affect the CD spectra and hence no significant conformational changes are induced upon formation of the ternary complexes. No changes in the CPL spectrum of NADH bound to lactate dehydrogenase were observed upon adding oxalate to form the ternary complex. Marked differences were found between the CPL spectra of binary and ternary complexes with liver alcohol dehydrogenase, while the CD spectra of these complexes were identical. It is concluded that a conformational change of the excited NADH molecule occurs in the binary but not in the ternary complex involving LADH, thus indicating an increased rigidity of the latter complex.     

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.     

Identification of serine and histidine adducts in complexes of trypsin and trypsinogen with peptide and nonpeptide boronic acid inhibitors by 1H NMR spectroscopy.

We have previously shown, in 15N NMR studies of the enzyme's active site histidine residue, that boronic acid inhibitors can form two distinct types of complexes with alpha-lytic protease. Inhibitors that are structural analogs of good alpha-lytic protease substrates form transition-state-like tetrahedral complexes with the active site serine whereas those that are not form complexes in which N epsilon 2 of the active site histidine is covalently bonded to the boron of the inhibitor. This study also demonstrated that the serine and histidine adduct complexes exhibit quite distinctive and characteristic low-field 1H NMR spectra [Bachovchin, W. W., Wong, W. Y. L., Farr-Jones, S., Shenvi, A. B., & Kettner, C. A. (1988) Biochemistry 27, 7689-7697]. Here we have used low-field 1H NMR diagnostically for a series of boronic acid inhibitor complexes of trypsin and trypsinogen. The results show that H-D-Val-Leu-boroArg and Ac-Gly-boroArg, analogs of good trypsin substrates, form transition-state-like serine adducts with trypsin, whereas the nonsubstrate analog inhibitors boric acid, methane boronic acid, butane boronic acid, and triethanolamine borate all form histidine adducts, thereby paralleling the previous results obtained with alpha-lytic protease. However, with trypsinogen, Ac-Gly-boroArg forms predominantly a histidine adduct while H-D-Val-Leu-boroArg forms both histidine and serine adducts, with the histidine adduct predominating below pH 8.0 and the serine adduct predominating above pH 8.0.(ABSTRACT TRUNCATED AT 250 WORDS)     

Combined biotin-terpyridine systems: a new versatile bridge between biology, polymer science and metallo-supramolecular chemistry

Biotin, a well-known binding unit for the proteins avidin and streptavidin, was combined with the chelating ligand terpyridine via polymeric and nonpolymeric spacers. An omega-amino-functionalized terpyridyl-poly(ethylene glycol) was prepared and utilized for complex formation with iron(II), nickel(II), and ruthenium(II) ions. The biocompatibility of the complex formation was investigated in aqueous media. Moreover, biotin was functionalized with a methoxy-poly(ethylene glycol) as a model system. The compounds were characterized by UV/vis and NMR spectroscopy as well as MALDI-TOF mass spectrometry. The systems represent a new combination of strong noncovalent binding units from both biology and synthetic supramolecular chemistry.