Conformational changes resulting from the carbazoylation of bovine α-chymotrypsin by p-nitrophenyl N2-acetyl-N1-arylmethylcarbazates

Several (N²-acetyl-N¹-arylmethylcarbazoyl)-α-chymotrypsins with p-substituents in the N¹-arylmethyl group have been prepared. Measurements of (a) accessibility of tryptophyl residues to modification by 2-hydroxy-5-nitrobenzyl bromide, (b) intrinsic fluorescence spectra in the absence and presence of sodium dodecyl sulphate, (c) thermal perturbation spectra indicate that, in general, tryptophyl residues are less accessible to solvent than in the free enzyme and the modified enzymes are more stable than α-chymotrypsin to denaturation by heat or sodium dodecyl sulphate. N²-Acetyl-N¹-p-dimethylaminobenzylcarbazoyl-α-chymotrypsin, however, contains more accessible tryptophyl residues than the other derivatives and is thermally less stable although it is more stable than the free enzyme.     

Structure of yeast hexokinase: IV. Low-resolution structure of enzyme—Substrate complexes revealing negative co-operativity and allosteric interactions

We conclude from X-ray diffraction studies at low resolution (7 Å) that the binding of sugar and nucleotide substrates to dimeric yeast hexokinase BII crystals exhibits both negative co-operativity and positive allosteric co-operativity. Difference electron density maps show the positions of sugar and nucleotide binding sites and extensive substrate-induced structural changes in the protein. Sugar substrates and inhibitors bind in the deep cleft that divides each subunit into two lobes and nucleotide substrates bind nearby to one site per dimer, which lies between the subunits and on the molecular symmetry axis. Although the inhibitors o- and p-iodobenzoylglucosamine and o-toluoylglucosamine bind equally to both subunits, the degree of substitution of glucose or xylose is very different for the two subunits. The substrate analog β, γ-imido ATP shows only one strong binding site per dimer. This negative co-operativity in substrate binding may result from the heterologous or non-equivalent association of the two subunits (Anderson et al., 1974), which provides non-equivalent environments for the two chemically identical subunits.Further, there is a positive allosteric interaction between the sugar and nucleotide binding sites. Sugar binding is required for nucleotide binding at the intersubunit site and the binding of nucleotide modifies the binding of sugars. These positive heterotropic interactions appear to be mediated by extensive substrate-induced structural changes in the enzyme.     

Protein immobilization on poly-(N-substituted) acrylamides

A series of water-soluble polymers containing side chains derived from N-acryloyl-β-alanine, N-ethylacrylamide and N-[3-(N′,N′,N′-trimethylammonio)propyl] acylamide chloride has been prepared and characterized. A related series of insoluble gels was also prepared. Protein may be attached to these materials by means of amide bond formation between carboxyl groups on the polymers and amino groups of the protein; the preparation and characterization of conjugates formed with α-chymotrypsin are described. Polymers bearing negatively or positively charged side chains are attached to this enzyme at only a single amino acid and the integrity of the active site is largely preserved in these systems. The corresponding gels are not able to bind as much enzyme as are the soluble polymers and bound enzyme is less active in these cases.     

The calcium ion binding site in bovine chymotrypsin A

The effect of Gd³⁺ on the nuclear magnetic resonance (nmr) relaxation rates, T_1m^?1 and T_2m^?1, of inhibitor protons in metal-inhibitor-α-chymotrypsin ternary complexes has been measured. The Solomon-Bloembergen equations were used to calculate the distance from the methyl protons of p-toluamidine (a competitive inhibitor) to the Gd³⁺ binding site which is 9.2 ± 0.5 Å. Calcium ion and gadolinium ion compete for the same binding site on α-chymotrypsin. Distances from the specificity pocket of α-chymotrypsin to the metal binding site have been measured by fluorescence energy transfer experiments. By observing energy transfer between proflavine and Nd³⁺, Pr³⁺, or Ho³⁺, we have been able to calculate a distance of approximately 10 Å between the two chromophores. This agrees well with the data obtained by nmr techniques and also gives nearly identical values to those obtained for trypsin (Darnall, D., Abbott, F., Gomez, J. E., and Birnbaum, E. R., Biochemistry15, 5017, 1976). This is consistent with the calcium ion binding sites being composed of the same residues in both trypsin and α-chymotrypsin.     

Comparison of the active-site conformations of bovine α-thrombin and meizothrombin(desF1) by electron spin resonance

The active site of the prothrombin activation intermediate meizothrombin(desF1) was probed using several fluorosulfonylphenyl spin labels specific for the active serine hydroxyl of serine proteases. The mobilities of the thrombin species inhibited with the nitroxide spin labelsm-IV [4-(2,2,6,6-tetramethyl-piperidine-1-oxyl)-m-(fluorosulfonyl)benzamide] andm-V [3-(2,2,5,5-tetramethyl-pyrrolidine-1-oxyl)-m-(fluorosulfonyl)benzamide], which are sensitive to differences betweenα- andγ-thrombin, were quite similar to that ofα-thrombin. That is, no major conformational differences between meizothrombin(desF1) andα-thrombin were observed in this region of the extended active site. On the other hand,p-IV [4-(2,2,6,6-tetramethyl piperidine-1-oxyl)-p-(fluorosulfonyl)benzamide],p-V [3-(2,2,5,5-tetramethylpyrrolidine-1-oxyl)-p-(fluorosulfonyl)benzamide], andm-VII [N-[m-(fluorosulfonyl)phenyl]-4-N-(2,2,6,6-tetramethyl-piperidine-1-oxyl)urea], which probe an apolar binding region of bovine thrombin, exhibited large differences in mobility betweenα-thrombin and meizothrombin(desF1). The conformational consequences of indole binding to spin-labeled thrombin species demonstrated that both species also possess an indole-binding site. However, the nitroxide mobility changes upon indole binding to the spin-labeled protein derivative were somewhat different between the two thrombin species under study. In addition, the effects of the benzamidine binding were quite similar for each labeled protein. Thus is appears that, while both species posses a fully functional active site, the region in meizothrombin(desF1) probed by spin labelsp-IV,p-V, andm-VII, which corresponds to the apolar binding region, differs in conformation fromα-thrombin.     

p-dimethylaminocinnamaldehyde derivatization for colorimetric detection and HPLC-UV/vis-MS/MS identification of indoles

Cytochrome P450 2A13 (CYP2A13) is a lung specific enzyme known to activate the potent tobacco procarcinogen 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK) into two carcinogenic metabolites. CYP2A13 has been crystallized and X-ray diffraction experiments illuminated the structure of this enzyme, but with an unknown ligand present in the enzyme active site. This unknown ligand was suspected to be indole but a selective method had to be developed to differentiate among indole and its metabolites in the protein sample. We successfully modified a microbiological colorimetric assay to spectrophotometrically differentiate between indole and a number of possible indole metabolites in nanomolar concentrations by derivatization with p-dimethylaminocinnamaldehyde (DMACA). Further differentiation of indoles was made by mass spectrometry (HPLC-UV/vis-MS/MS) utilizing the chromophore generated in the DMACA conjugation as a UV signature for HPLC detection. The ligand in the crystallized protein was identified as unsubstituted indole, which facilitated refinement of two alternate conformations in the CYP2A13 crystal structure active site.     

Interactions of five- and six-membered cyclic esters with α-chymotrypsin: Kinetic evidence for covalent enzyme intermediates

Interactions of α-chymotrypsin with 2-coumaranone (I), 3,4-dihydrocoumarin (II), o-hydroxy-α-toluenesulfonic acid sultone (III), and β-o-hydroxyphenylethanesulfonic acid sultone (IV) were studied in the presence of 14% acetonitrile at pH 7.0 by means of the proflavin displacement technique and by inhibition of N-acetyl-l-tryptophan ethyl ester (ATrEE) hydrolysis. Under saturating conditions of either I, II, or III, an enzyme intermediate was shown to accumulate using either the proflavin displacement technique or the ATrEE activity assay. The intermediates have characteristics of covalent enzyme-substrate compounds and are believed to decompose simultaneously by two pathways, one to give free enzyme and hydrolyzed cyclic ester, and the other to give the original cyclic ester and free enzyme. With α-chymotrypsin and III the observed first-order rate constant for decomposition of the intermediate by the two pathways was 0.19 ± 0.04 min^?1, while the rate constant for the hydrolytic pathway alone was 0.013 ± 0.0009 min^?1. These results indicate that the covalent-like intermediate with this sultone is not only capable of reverting to starting cyclic ester but prefers this pathway over hydrolysis. Sultone IV was found to bind to enzyme; but in contrast to the behavior of esters I–III, the binding did not result in accumulation of a covalent-like intermediate.     

Occurrence of an oxalate oxidase in Sorghum leaves

An oxalate oxidase found in the 15 000 g supernatant of 10-day-old sorghum leaves exhibited a pH optimum of 5 and a temperature optimum of 45° and was unaffected by Na⁺. The enzyme activity remained linear up to 10 min and the apparent K_m for oxalate was 2.4 × 10^?5 M. The enzyme activity was strongly inhibited by sodium dithionite and α,α′-dipyridyl. Inhibition by the latter was specifically reversed by Fe²⁺. The activity of the dialysed enzyme was restored by the addition of Fe²⁺ and FAD. Inhibition of the enzyme by iodoacetate, p-chloromercuribenzoate and N-methylmaleimide revealed that SH groups at the active site are essential.     

Reactive sulfhydryl groups of coenzyme B12-dependent diol dehydrase: Differential modification of essential and nonessential ones

The apoenzyme of diol dehydrase was inactivated by four sulfhydryl-modifying reagents, p-chloromercuribenzoate, 5,5′-dithiobis(2-nitrobenzoate) (DTNB), iodoacetamide, and N-ethylmaleimide. In each case pseudo-first-order kinetics was observed. p-Chloromercuribenzoate modified two sulfhydryl groups per enzyme molecule and modification of the first one resulted in complete inactivation of the enzyme. DTNB also modified two sulfhydryl groups, but modification of the second one essentially corresponded to the inactivation. In both cases, the inactivation was reversed by incubation with dithiothreitol. Cyanocobalamin, a potent competitive inhibitor of adenosylcobalamin, protected the essential residue, but not the nonessential one, against the modification by these reagents. By resolving the sulfhydryl-modified cyanocobalamin-enzyme complex, the enzyme activity was recovered, irrespective of treatment with dithiothreitol. From these results, we can conclude that diol dehydrase has two reactive sulfhydryl groups, one of which is essential for catalytic activity and located at or in close proximity to the coenzyme binding site. The other is nonessential for activity. Neitherp-chloromercuribenzoate- nor DTNB-modified apoenzyme was able to bind cyanocobalamin, whereas the iodoacetamide- and N-ethylmaleimide-modified apoenzyme only partially lost the ability to bind cyanocobalamin. The inactivation of diol dehydrase by p-chloromercuribenzoate and DTNB did not bring about dissociation of the enzyme into subunits. Total number of the sulfhydryl groups of this enzyme was 14 when determined in the presence of 6 m guanidine hydrochloride. No disulfide bond was detected.     

Substrate positions and induced-fit in crystalline adenylate kinase.

The binding positions of ATP and AMP in pig muscle adenylate kinase (EC 2.7.4.3) have been located by X-ray diffraction analysis. For this purpose crystals have been soaked with solutions containing substrates and substrate analogues. Two adenosine pockets and the region of the phosphates have been identified. In combination with other experimental data the pockets have been assigned to the AMP site and the ATP site, respectively. Moreover, the results suggest that the known conformations of adenylate kinase reflect an induced-fit of the enzyme: conformation B being related to the free enzyme E and conformation A being related to E¹, the enzyme species after a substrate-induced conformational change.     

Vanadium containing bromoperoxidase - Insights into the enzymatic mechanism using X-ray crystallography

The X-ray crystal structure of the vanadium bromoperoxidase from the red algae Corallina pilulifera has been solved in the presence of the known substrates, phenol red and phloroglucinol. A putative substrate binding site has been observed in the active site channel of the enzyme. In addition bromide has been soaked into the crystals and it has been shown to bind unambiguously within the enzyme active site by using the technique of single anomalous dispersion. A specific leucine amino acid is seen to move towards the bromide ion in the wild-type enzyme to produce a hydrophobic environment within the active site. A mutant of the enzyme where arginine 397 has been changed to tryptophan, shows a different behaviour on bromide binding. These results have increased our understanding of the mechanism of the vanadium bromoperoxidases and have demonstrated that the substrate and bromide are specifically bound to the enzyme active site.     

Modification of the acceptor binding site of gamma-glutamyl transpeptidase by the diazonium derivatives of p-aminohippurate and p-aminobenzoate

Hippurate and maleate have been shown to bind to the aminoacylglycine (acceptor) binding site of γ-glutamyl transpeptidase, thereby stimulating the hydrolysis of γ-glutamyl compounds at the expense of transpeptidation (Thompson, G. A., and Meister, A. (1979) J. Biol. Chem.254, 2956–2960; Thompson, G. A., and Meister, A. (1980) J. Biol. Chem.255, 2109–2113). It has now been found that a number of benzoate derivatives also bind and modulate rat kidney transpeptidase, as indicated by their ability to enhance the rate of inactivation of transpeptidase by the glutamine antagonist l-(αS, 5S)-α-amino-3-chloro-4,5-dihydro-5-isoxazoleacetic acid (AT-125). Furthermore, rapid loss of transpeptidase activity results upon preincubation of the enzyme with the diazonium derivatives of p-aminohippurate and p-aminobenzoate. The modified enzyme can still hydrolyze γ-glutamyl substrates but is no longer modulated by hippurate and maleate. Loss of transpeptidase activity was not associated with incorporation of radioactive label from diazotized [¹⁴C]p-aminohippurate. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the modified enzyme revealed a nondissociable species, M_r 68,000, shown to result from crosslinking of the two subunits of transpeptidase (M_r 46,000 and 22,000, respectively). The crosslinking of the subunits paralleled the extent of inactivation of transpeptidation activity and both crosslinking and inactivation were prevented by treatment with the diazotized derivatives in the presence of either hippurate or maleate. These and other data indicate that the diazonium derivatives of p-aminohippurate and p-aminobenzoate interact with the acceptor binding site and produce a stable bond between amino acid residues in the vicinity of this site which, thus, appears to be located in the intersubunit contact region.     

The binding of pentaammineruthenium (III) to RNase B and RNase A+d(pA)4 in the crystalline state

The binding of pentaammineruthenium (III) to ribonuclease A and B both free and complexed with d(pA)₄ has been examined in the crystalline state through the application of X-ray diffraction and difference Fourier techniques. In crystals of native RNase B, the reagent was observed to have many binding sites, some entirely electrostatic in nature and others consistent with coordination to histidine residues. The primary histidine in the latter case was 105 with 119 also partially substituted. In crystals of RNase A+d(pA)₄ complex only a single, extremely strong site of substitution was observed, and this was 2.4 Å from the native position of the imidazole ring of histidine 105. Thus, the results of these X-ray diffraction studies appear to be quite consistent with the findings of earlier NMR studies and with the results obtained in crystals of the gene 5 DNA binding protein.     

5.5 Å crystallographic structure of penicillin β-lactamase and radius of gyration in solution

An electron density map of crystalline R-TEM Escherichia coli β-lactamase (penicillinase) has been calculated from X-ray diffraction data at 5.5 Å resolution with protein phases based on Friedel mates from a high-quality samarium derivative. The mean figure of merit for 854 independent reflections is 0.75. The monomeric molecule is slightly ellipsoidal and contains one and possibly two regions of α-helix which are 25 Å long. The Crystallographic search for the substrate binding site has so far been inconclusive. The radius of gyration of the enzyme in solution at pH 7 is 17.1 ± 1.0 Å from small-angle X-ray scattering measurements. This compares with 18.6 å calculated from the low-resolution electron density map of the molecule in the crystal.     

Effect of hydroxynitrobenzylation of tryptophan-177 on reactivity of active site cysteine-25 in papain

It is known that the enzymatic activity of papain (EC 3.4.22.2) toward α-N-benzoyl-l-arginine p-nitroanilide can be substantially increased by hydroxynitrobenzylation of Trp-177 through reaction of the enzyme with the active site-directed reagent, 2-chloromethyl-4-nitrophenyl (N-carbobenzoxy)glycinate (S.-M. T. Chang and H. R. Horton, 1979, Biochemistry18, 1559–1563). To determine the effect of such hydroxynitrobenzylation on the nucleophilicity of the essential thiol group at the active site of the enzyme, rates of inactivation by S_N2 reactions of Cys-25 with chloroacetamide and chloroacetate and by Michael addition of Cys-25 to N-ethylmaleimide were monitored. The kinetics revealed that, at pH 6.5, the reactivities of the sulfhydryl group of hydroxynitrobenzylated papain with chloroacetamide and with N-ethylmaleimide are 24 and 27% greater than those of the sulfhydryl group of native papain. At pH 7.1, the rate enhancements are 34 and 39%, respectively. These increases in reactivity of Cys-25 as an attacking nucleophile appear to account for the increased catalytic activity of hydroxnitrobenzyl-papain toward an oligopeptide substrate, α-N-benzoyl-l-phenylalanyl-l-valyl-l-arginine p-nitroanilide, and toward an ester substrate, N-carbobenzoxyglycine p-nitrophenyl ester. However, the presence of the hydroxynitrobenzyl reporter group provides substantially greater improvement (250%) in enzymatic efficiency toward α-N-benzoyl-l-arginine p-nitroanilide, apparently by blocking nonproductive binding of this substrate to the enzyme. Fluorescence changes accompanying the various chemical modifications are interpreted in terms of a charge-transfer interaction between the imidazolium ion of His-159 and the indole moiety of Trp-177 in the active form of native papain, which should help to stabilize the catalytically essential mercaptide-imidazolium ion-pair (Cys-25, His-159).     

Fluorine nmr studies of poly(N-acryloyl-β-alanine)–α-chymotrypsin conjugates

Fluorine nmr experiments carried out at 51.0 and 94.1 MHz have been used to explore the interaction of the probe molecule p-fluorocinnamate with conjugates formed from α-chymotrypsin and poly(N-acryloyl-β-alanine). The data obtained include enzyme-induced chemical-shift effects, spin-lattice (R₁) and transverse (R₂) relaxation rates, and the rate constant for dissociation of the fluorocinnamate–enzyme complexes. Analysis of the results indicates that while overall molecular tumbling of the enzyme molecule is not greatly changed by attachment of polymers of various sizes, conjugated polymer can appreciably affect the structure of the p-fluorocinnamate binding site. The important variable involved in such structural changes appears to be the amount of polymer present per mole of protein.     

Characterization of the extended carbohydrate binding site of concanavalin A: Specificity for interaction with the nonreducing termini of α-(1→2)-linked disaccharides

p-Nitrophenyl 2-O-α-d-galactopyranosyl-α-d-mannopyranoside and p-nitrophenyl 2-O-α-d-glucopyranosyl-α-d-mannopyranoside were synthesized and the interactions of these disaccharides with concanavalin A (con A) were characterized. The kinetics of binding of the galactopyranosyl-containing disaccharide to con A were found to be similar to those observed with monosaccharides in that monophasic time dependencies for binding were observed. The glucopyranosyl-containing disaccharide, however, exhibited biphasic time dependencies which were similar to those previously observed for the binding of p-nitrophenyl 2-O-α-d-mannopyranosyl-α-d-mannopyranoside to con A. These results support a model wherein the α-(1→2)-linked disaccharides which exhibit biphasic binding kinetics must be able to bind to con A in two different and mutually exclusive orientations. The ability to bind to con A in two orientations is shared by α-(1→2)-linked disaccharides in which both glycosyl residues can interact separately with the primary glycosyl binding site of con A. According to the model, the initial fast phase of the biphasic reaction reflects binding of the ligand in two orientations so that two complexes are formed in amounts determined by the relative values of the rate constants for formation of each complex. The subsequent slow phase is proposed to reflect a slow equilibration of the less stable complex to the thermodynamically more stable one. In the more stable complex, the glycosyl residue at the reducing end of the disaccharide occupies the primary glycosyl binding site. The added stability of this complex is attributed to extended interactions between con A and groups on the second glycosyl residue. An axial orientation of OH-2 of the second glycopyranosyl residue appears to be the most important determinant for the extended interaction.     

Interaction of α-Chymotrypsin with Dimethyl Sulfoxide: A Change of Substrate Could “Change” the Interaction Mechanism

The kinetic behavior of -chymotrypsin was studied in water–DMSO mixtures at concentrations of the organic solvent that do not cause irreversible denaturation of the enzyme. Various substrates (N-substituted derivatives of L-tyrosine) were found to display substantially different kinetic patterns of interaction with -chymotrypsin, which can be described by totally different kinetic schemes. The differences were ascribed to competition between the N-acyl group of the substrate and the DMSO molecule at the S ₂ site of substrate binding to the active site of the enzyme.     

Quantitative affinity chromatography of alpha-chymotrypsin

Affinity chromatography on a column of 4-phenylbutylamine, immobilized on succinylated polyacrylic hydrazide agarose, has been employed to study binding of ligands to α-chymotrypsin. In contrast to earlier studies of competitive elution phenomena, where an added soluble ligand interferes with enzyme binding to an affinity matrix, benzyloxycarbonyl derivatives of aromatic acids have been shown to facilitate binding of chymotrypsin to this matrix. This behavior has been analyzed in terms of an expanded binding scheme for affinity chromatography including the formation of a ternary complex (α-chymotrypsin · benzyloxycarbonyl-amino acid · 4-phenylbutylamine · matrix) where the soluble ligand and immobilized ligand bind to different sites. Equations to describe the phenonema have been derived and utilized to quantitate equilibrium constants for dissociation of the binary and ternary complexes. Benzyloxycarbonyl-Ala-Ala was found to promote earlier elution of the enzyme from its affinity matrix. Other ligands known to bind to the active site do not alter the binding to the 4-phenylbutylamine affinity matrix. These results illustrate the conclusion that binding of a small molecule can alter affinity retention in positive, negative, or neutral modes. This suggests that affinity chromatography could be “fine-tuned” by appropriate selection of cosolutes and illustrates the value of relatively weakly binding affinity matrices in enzyme studies.     

Self-assembled peptide fibers from valylvaline bola-amphiphiles by a parallel β-sheet network

A series of dipeptide-based bola-amphiphiles, bis(N-α-amide-L-valyl-L-valine) 1, n-alkane dicarboxylate (n=4–12), have been synthesized. The bola-amphiphiles with n=4 and 6 self-assembled to form crystalline solids in water, whereas those with n=7–12 produced peptide fibers. FT-IR spectroscopy and X-ray diffraction patterns revealed that the peptide fibers have parallel-type β-sheet networks between the valylvaline units. FT-IR deconvolution study of carboxyl regions indicated that these crystalline solids and peptide fibers are stabilized by interlayer bifurcated and intralayer lateral hydrogen-bond networks between the end carboxylic acid groups, respectively.