Triplet state of tryptophan in proteins. 2. Differentiation between tryptophan residues 62 and 108 in lysozyme.

We have used optically detected magnetic resonance (ODMR) to characterize the degree of solvent availability of the tryptophan residues in lysozyme that are likely to be responsible for the observed phosphorescence. From the phosphorescence spectra, ODMR zero-field splittings (zfs), and ODMR line widths, we concur with the X-ray structure [Blake, C. C., Mair, G. A., North, A. C. T., Phillips, D. C., & Sarma, V. R. (1967) Proc. R. Soc. London, ser. B 167, 365-377] that Trp-62 behaves as an exposed residue and Trp-108 is buried. In addition, we present evidence that ODMR can be used in conjunction with conventional phosphorescence to evaluate the degree of order in the microenvironments of tryptophan in a protein containing several tryptophans. By the specific modification of residues Trp-62 and Trp-108, we have identified those portions of the ODMR lines in the native enzyme that are due to those specific residues. Barring major enzyme conformational changes in the vicinity of unmodified tryptophan residues when Trp-62 or Trp-108 are selectively modified, we find that Trp-108 dominates both the phosphorescence and the ODMR signals in native lysozyme. The results are discussed in view of previous fluorescence findings.     

Multiple role of hydrophobicity of tryptophan-108 in chicken lysozyme: structural stability, saccharide binding ability, and abnormal pKa of glutamic acid-35.

Trp108 of chicken lysozyme is in van der Waals contact with Glu35, one of two catalytic carboxyl groups. The role of Trp108 in lysozyme function and stability was investigated by using mutant lysozymes secreted from yeast. By the replacement of Trp108 with less hydrophobic residues, Tyr (W108Y lysozyme) and Gln (W108Q lysozyme), the activity, saccharide binding ability, stability, and pKa of Glu35 were all decreased with a decrease in the hydrophobicity of residue 108. Namely, at pH 5.5 and 40 degrees C, the activities of W108Y and W108Q lysozymes against glycol chitin were 17.3 and 1.6% of that of wild-type lysozyme, and their dissociation constants for the binding of a trimer of N-acetyl-D-glucosamine were 7.4 and 309 times larger than that of wild-type lysozyme, respectively. For the reversible unfolding at pH 3.5 and 30 degrees C, W108Y and W108Q lysozymes were less stable than wild-type lysozyme by 1.4 and 3.6 kcal/mol, respectively. As for the pKa of Glu35, the values for W108Y and W108Q lysozymes were found to be lower than that for wild-type lysozyme by 0.2 and by 0.6 pKa unit, respectively. The pKa of Glu35 in lysozyme was also decreased from 6.1 to 5.4 by the presence of 1-3 M guanidine hydrochloride, or to 5.5 by the substitution of Asn for Asp52, another catalytic carboxyl group. Thus, both the hydrophobicity of Trp108 and the electrostatic interaction with Asp52 are equally responsible for the abnormally high pKa (6.1) of Glu35, compared with that (4.4) of a normal glutamic acid residue.(ABSTRACT TRUNCATED AT 250 WORDS)     

Binding of substrate analogs to hen egg-white lysozyme with an ester linkage between Glu 35 and Trp 108.

The interactions of the substrate analogs beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which an ester linkage had been formed between Glu 35 and Trp 108 (108 ester lysozyme), were studied by the circular dichroic and fluorescence techniques, and were compared with those for intact lysozyme. The binding constants of beta-methyl-GlcNAc and (GlcNAc)2 to 108 ester lysozyme were essentially the same as those for intact lysozyme in the pH range of 1 to 5. Above pH 5, the binding constants of these saccharides to 108 ester lysozyme did not change with pH, while the binding constants to intact lysozyme decreased. This indicates that Glu 35 (pK 6.0 in intact lysozyme) participates in the binding of these saccharides. The extent and direction of the pK shifts of Asp 52 (pK 3.5), Asp 48 (pK 4.4), and Asp 66 (pK 1.3) observed when beta-methyl-GlcNAc is bound to 108 ester lysozyme were the same as those for intact lysozyme. The participation of Asp 101 and Asp 66 in the binding of (GlcNAc)2 to 108 ester lysozyme was also the same as that for intact lysozyme. These findings indicate that the conformations of subsites B and C are not changed by the formation of the ester linkage. On the other hand, the binding constants of (GlcNAc)3 to 108 ester lysozyme were higher than those for intact lysozyme at all pH values studied. This result is interpreted in terms of an increase in the affinity for a GlcNAc residue of subsite D, which is situated near the esterified Glu 35.     

Stopped-flow chemical modification with N-bromosuccinimide: a good probe for changes in the microenvironment of the Trp 62 residue of chicken egg white lysozyme

The stopped-flow chemical modification with N-bromosuccinimide (NBS) of Trp 62 of hen (chicken) egg white lysozyme (EC 3.2.1.17) was found to depend greatly on pH: it was not observed at pH's above 7, but it was observed at pH's lower than 6. In addition, at pH's between 6 and 7 the NBS modification showed a delta epsilon pH profile similar to a "titration curve," giving a pK (congruent to 6.5) nearly equal to the pK (congruent to 6.2) of a catalytic residue, Glu 35. The stopped-flow chemical (NBS) modification of N-acetyl-L-tryptophan ethyl ester, a model compound of Trp 62, does not depend on pH at the pH's examined, approximately 3.5-8.5. These experimental results suggest that a change in the state of Trp 62 at Subsite C is induced by protonation-deprotonation of an ionizable residue, which could be Glu 35 (catalytic site), indicating that stopped-flow NBS modification is a good probe for detection of changes in the micorenvironment around the tryptophan residue(s) of enzymes.     

The tryptophan residues of mitochondrial creatine kinase: roles of Trp-223, Trp-206, and Trp-264 in active-site and quaternary structure formation.

The 5 tryptophan residues of chicken sarcomeric mitochondrial creatine kinase (Mib-CK) were individually replaced by phenylalanine or cysteine using site-directed mutagenesis. The mutant proteins were analyzed by enzyme kinetics, fluorescence spectroscopy, circular dichroism, and conformational stability studies. In the present work, Trp-223 is identified as an active-site residue whose replacement even by phenylalanine resulted in > or = 96% inactivation of the enzyme. Trp-223 is responsible for a strong (18-21%) fluorescence quenching effect occurring upon formation of a transition state-analogue complex (TSAC;Mib-CK.creatine.MgADP.NO3-), and Trp-223 is probably required for the conformational change leading to the TSAC-induced octamer dissociation of Mib-CK. Replacement of Trp-206 by cysteine led to a destabilization of the active-site structure, solvent exposure of Trp-223, and to the dissociation of the Mib-CK dimers into monomers. However, this dimer dissociation was counteracted by TSAC formation or the presence of ADP alone. Trp-264 is shown to be located at the dimer-dimer interfaces within the Mib-CK octamer, being the origin of another strong (25%) fluorescence quenching effect, which was observed upon the TSAC-induced octamer dissociation. Substitution of Trp-264 by cysteine drastically accelerated the TSAC-induced dissociation and destabilized the octameric structure by one-fourth of the total free interaction energy, probably by weakening hydrophobic contacts. The roles of the other 2 tryptophan residues, Trp-213 and Trp-268, could be less well assigned.     

Preparation and characterization of an active lysozyme derivative: Kyn 62-lysozyme.

A novel method for the preparation of Kyn 62-lysozyme, in which tryptophan 62 is replaced by kynurenine, is reported. Hen egg-white lysozyme was ozonized in aqueous solution to yield one N'-formylkynurenine residue and deformylated with hydrochloric acid in frozen solution at -10 degrees C. Crude Kyn 62-lysozyme was purified by affinity and Bio Rex 70 chromatography successively. Kyn 62-lysozyme retains affinity for chitin and is essentially an active enzyme with a slightly weakened but distinct catalytic activity. After this modification, the enzyme activity was changed differently depending on the kind of substrate. At the individual optimum pH's, lytic activity was largely retained (80% active), but the catalytic efficiency for hydrolyzing glycol chitin was relatively low (30% active). Lysis of M. lysodeikticus cell suspensions was optimally catalyzed by Kyn 62-lysozyme at pH 6.2 and at 0.088 ionic strength. These values are lower by 1.3 pH unit and 0.04 ionic strength, respectively, than those of intact lysozyme. The optimum pH and ionic strength for the hydrolysis of neutral substrates were scarcely affected. These results suggest the significance of electrostatic interaction in the lysis of lysozyme. Relatively limited loss of activity induced by modification of the 62nd residue, which is thought to participate directly in the binding of the substrate at subsite C, is discussed on the basis of the similarity of side chain structure in tryptophan and kynurenine.     

Ni2+ binds to active site of hen egg-white lysozyme and quenches fluorescence of Trp62 and Trp108

We found that the maximum emission of the tryptophyl fluorescence of hen egg-white lysozyme is shifted from 337 to 323 nm and quenched to the extent of 55% with an increase in concentrations of NiCl2 from 0 to 2M in 50 mM Na acetate buffer (pH 4.7). In contrast, NaCl does not influence the fluorescence of lysozyme up to 2M. To elucidate the particular effects of Ni2+ on the tryptophyl fluorescence of lysozyme, we have measured the assembly behavior and secondary structure of lysozyme in various concentrations of NiCl2, and determined the structures of lysozyme crystals grown in 0.3, 0.5, and 1.0M NiCl2, respectively. The results of analytical centrifugation and circular dichroism experiments show that lysozyme keeps a monomer state and has an identical secondary structure, irrespective of NiCl2 concentrations. The crystal structures show that all crystals grown in different concentrations of NiCl2 have an identical main chain and side chain conformation. And one Ni2+ binding with Odelta atom of Asp52 in the active site and coordinating with five water molecules to form hexagonal coordination has been determined for each crystal structure. Based on these results, we have proposed that Ni2+ quenches the fluorescence of Trp62 and Trp108 due to the binding of Ni2+ to the active site of lysozyme.     

Persistence of external chloride and DIDS binding after chemical modification of Glu-681 in human band 3

Although its primary function is monovalent anion exchange, theband 3 protein also cotransports divalent anions together with protonsat low pH. The putative proton binding site, Glu-681 in humanerythrocyte band 3, is conserved throughout the anion exchanger family(AE family). To determine whether or not the monovalent anion bindingsite is located near Glu-681, we modified this residue with Woodward'sreagent K(N-ethyl-5-phenylisoxazolium-3'-sulfonate; WRK). Measurements ofCl binding by³⁵Cl-NMR show that externalCl binds to band 3 evenwhen Cl transport isinhibited ~95% by WRK modification of Glu-681. This indicates thatthe external Cl bindingsite is not located near Glu-681 and thus presumably is distant fromthe proton binding site. DIDS inhibitsCl binding even when WRK isbound to Glu-681, indicating that the DIDS binding site is also distantfrom Glu-681. Our data suggest that the DIDS site and probably also theexternally facing Cltransport site are located nearer to the external surface of themembrane than Glu-681.

     

Role of Asp-9 and Glu-36 in the active site of the pneumococcal CPL1 lysozyme: an evolutionary perspective of lysozyme mechanism.

The role of carboxylic amino acids Asp-9 and Glu-36 in the activity of CPL1 lysozyme was investigated by site-directed mutagenesis. The enzymatic activity of the single mutants D9E, D9N, D9H, D9K, D9A, E36D, E36Q, E36K, and E36A and of the double mutant D9A-E36A was analyzed using a highly sensitive radioactive assay. All mutants but D6K showed detectable activities. Interestingly, the mutants E36D and E36Q retained 67% and 37% activity, respectively. Amino acid replacements at position 9 turned out to be more critical for activity than at position 36. In analogy to the mechanism described for hen egg-white lysozyme, where the proton donor play a central role, we propose that, in the CPL1 lysozyme, Asp-9 might act as the proton donor for activation of the substrate, and Glu-36 could help in the stabilization of the intermediate oxocarbocation. The residual activity of lysozyme mutants lacking one or two of the acidic amino acids may be explained by the participation of a water molecule as proton donor and/or to electrostatic contributions in the active center stabilizing the transition state of the reaction. Our results are in agreement with the hypothesis that enzymes have been optimized during evolution from an ancestral protein able to bind more tightly the transition state of the substrate than the substrate itself, by the acquisition of amino acids serving a function in catalysis.     

Bacillus thuringiensis crystal protein: effect of chemical modification of the cysteine and lysine residues.

The 16 cysteine residues of reduced protoxin from Bacillus thuringiensis subsp. kurstaki HD-73 can be quantitatively reacted with: (a) iodoacetic acid, to give carboxymethyl protoxin; (b) iodoacetamide, giving carbaminomethyl protoxin and (c) N-(beta-iodoethyl)trifluoroacetamide to give aminoethyl protoxin. The carboxymethyl derivative was found to be significantly more soluble at neutral pH values where both the native protoxin and the carbaminomethyl derivative exhibit low solubilities. At the alkaline pH values (pH 9.5-10.5) normally used to solubilize the crystal protein, the native protein was slightly more soluble than either the carboxymethyl or the carbaminomethyl derivatives. The aminoethyl derivative had an extremely low solubility at all pH values. Succinic anhydride reacted with only 35% of the lysine residues in both the carboxymethyl and the carbaminomethyl protoxin derivatives. Nonetheless, these succinylated protoxins exhibited significantly increased solubilities at neutral pH values. All the derivatives were found to retain full insecticidal activity toward spruce budworm (Choristeneura fufimerana) larvae. It is concluded that all the cysteine residues and modified lysine residues are on the surface of the protein and that derivatization does not alter the conformation of the solubilized protoxin.     

Human leukocyte cathepsin G. Subsite mapping with 4-nitroanilides, chemical modification, and effect of possible cofactors

The extended substrate binding site of cathepsin G from human leukocytes has been mapped by using a series of peptide 4-nitroanilide substrates. The enzyme has a significant preference for substrates with a P1 Phe over those with the other aromatic amino acids Tyr and Trp. The S2 subsite was mapped with the substrates Suc-Phe-AA-Phe-NA where AA was 13 of the 20 amino acid residues commonly found in proteins. The best residues were Pro and Met. The S3 subsite was mapped with the sequence Suc-AA-Pro-Phe-NA by using 14 different amino acid residues for AA. The two best residues were the isosteric Val and Thr. No significant improvement in reactivity was obtained by extending the substrate to include seven different P4 residues. The kinetic parameters for cathepsin G are significantly slower than those for many other serine proteases. Changes in the reaction conditions and addition of possible cofactors or ligands were in general found to have little effect on the enzymatic activity, while chemical modifications and proteolysis destroyed the activity of cathepsin G. Cathepsin G hydrolyzed peptides containing model desmosine residues and prefers the hydrophobic picolinoyllysine derivative over lysine by substantial margins at both the S4 and S2 subsites but will not tolerate it at S3. Substrates with sequences related to the cathepsin G cleavage site in angiotensin I and angiotensinogen, and the reactive site of alpha 1-antichymotrypsin, were hydrolyzed effectively by enzyme, but with unexceptional rates. Our results indicate that the natural substrate(s) and function(s) of cathepsin G still remain to be discovered.     

Primary structure, physicochemical properties, and chemical modification of NAD(+)-dependent D-lactate dehydrogenase. Evidence for the presence of Arg-235, His-303, Tyr-101, and Trp-19 at or near the active site.

The NAD(+)-dependent D-lactate dehydrogenase was purified to apparent homogeneity from Lactobacillus bulgaricus and its complete amino acid sequence determined. Two gaps in the polypeptide chain (10 residues) were filled by the deduced amino acid sequence of the polymerase chain reaction amplified D-lactate dehydrogenase gene sequence. The enzyme is a dimer of identical subunits (specific activity 2800 +/- 100 units/min at 25 degrees C). Each subunit contains 332 amino acid residues; the calculated subunit M(r) being 36,831. Isoelectric focusing showed at least four protein bands between pH 4.0 and 4.7; the subunit M(r) of each subform is 36,000. The pH dependence of the kinetic parameters, Km, Vm, and kcat/Km, suggested an enzymic residue with a pKa value of about 7 to be involved in substrate binding as well as in the catalytic mechanism. Treatment of the enzyme with group-specific reagents 2,3-butanedione, diethylpyrocarbonate, tetranitromethane, or N-bromosuccinimide resulted in complete loss of enzyme activity. In each case, inactivation followed pseudo first-order kinetics. Inclusion of pyruvate and/or NADH reduced the inactivation rates manyfold, indicating the presence of arginine, histidine, tyrosine, and tryptophan residues at or near the active site. Spectral properties of chemically modified enzymes and analysis of kinetics of inactivation showed that the loss of enzyme activity was due to modification of a single arginine, histidine, tryptophan, or tyrosine residue. Peptide mapping in conjunction with peptide purification and amino acid sequence determination showed that Arg-235, His-303, Tyr-101, and Trp-19 were the sites of chemical modification. Arg-235 and His-303 are involved in the binding of 2-oxo acid substrate whereas other residues are involved in binding of the cofactor.     

Neocarzinostatin: effect of modification of side chain amino and carboxyl groups on chemical and biological properties.

The antitumor protein neocarzinostatin (NCS), isolated from Streptomyces carzinostaticus, is a single chain polypeptide with 109 amino acid residues. Complete acylation of the amino groups (alanine-1 and lysine-20) was observed when NCS was allowed to react with 3-(4-hydroxyphenyl)-propionic acid N-hydroxysuccinimide ester at pH 8.5. Since the ensuing bis[(alanine-1, lysine-20)-3-(4-hydroxyphenyl)]-propionamide NCS was fully active in antibacterial potency and in the inhibition of growth of leukemic (CCRF-CEM) cells in vitro, it appears that the two amino groups in the protein are not essential for biological activity. Radiolabeled NCS was prepared by using a tritiated or 125I-labeled acylating agent. Since the CD spectra of native and bis(alanine-1, lysine-20)-amino modified NCS were indistinguishable, there is presumably no change in the native conformation of the protein due to acylation. Reaction of NCS with ammonium chloride in the presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.75 converted all the 10 carboxyl groups into carboxamides and produced a protein derivative of basic character. This modification caused a change in the native conformation of the protein accompanied by a loss in biological inhibitory activities.     

Protein modification in nonaqueous media. Preparation and properties of matrix-supported lysozyme

Hen's egg white lysozyme (EC 3.2.1.17) has been covalently attached to a polystyrene matrix via interaction of protein nucleophiles with an aromatic imidazolide function under anhydrous conditions. The polymer-enzyme complex is prepared in a way which allows nonaqueous solubilization of the complex. The activity of the bound enzyme compares favorably with the activity of the native protein. The pH optima for the matrix-supported protein are shifted toward the basic side. The effect of substrate concentration on rate has been determined. (A preliminary report of this work has been published: G. J. Bartling, H. D. Brown, S. K. Chattopadhyay, Nature 243 , 342–344 (1973).)     

Surface modification of polymers: chemical, biological and surface analytical challenges

Surface modification methods can optimise the biocompatibility or the specificity of biointeraction of a biosensor or medical device. With only the surface modified, the manufacture and implantation protocol remain unchanged. This review article summarises some of the chemical, surface analytical and biological challenges associated with surface modification of biosensors and biomedical devices.     

Oxidation and chemical modification of lung beta-galactoside-specific lectin.

Galaptins are small, soluble, lectins with a specificity for beta-galactose residues. Many galaptins are inactivated by atmospheric oxygen and are protected by disulphide-reducing reagents. We find that each subunit of rat lung galaptin contains one residue of tryptophan and six of cysteine. Oxygen inactivates rat lung galaptin by oxidation of the cysteine residues. During oxidation, the normal dimeric structure is maintained and all disulphide bonds are formed within individual subunits. Exogenous thiols protect against inactivation, but oxidized thiols accelerate inactivation. Human lung fibroblast galaptin is almost completely inactivated within 1 h in tissue culture medium at 37 degrees C. Alkylation of native rat lung galaptin with iodoacetate or ethyleneimine causes substantial loss of activity. The dimeric galaptin structure is maintained. In contrast, alkylation with iodoacetamide yields carboxamidomethyl-galaptin, which is fully active and stable to atmospheric oxygen in the absence of disulphide-reducing reagents. This derivative is very useful for studies of galaptin properties and function.