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Biomedical subjects

F Sakiyama

Publications and source records attributed to F Sakiyama.

At least 109 records · Page 6Linked to original sources
PubMed1983

NMR titration studies of histidine 57 and the [methylene-13C]PMS group in the phenylmethanesulfonyl (PMS) derivative of Streptomyces erythraeus trypsin.

1H and 13C NMR titrations were performed on PMS-St. trypsin derived by PMSF modification of Ser 195 in Streptomyces erythraeus trypsin, which is devoid of auto-catalytic degradation activity at pH approximately 8. NMR titration of the imidazole C2 proton showed that His 57 had the pKa value of 6.9 in both native St. trypsin and PMS-St. trypsin, suggesting that the adjacent hydroxyl group of Ser 195 had no effect or a very weak effect on the acid-base properties of the imidazole ring in the catalytic triad. A small change in the chemical shift of the isotopically enriched methylene carbon in the PMS moiety of [methylene-13C]PMS-St. trypsin was observed between pH 6 and 8. The titration curve had an inflexion at pH 6.9 and the mode of transition was apparently sigmoidal, although a Hill coefficient of more than unity was suggested. It is thus likely that His 57 is responsible for this transition. Based on these results, the role of His 57 in the catalytic triad of the active site in serine protease is discussed.

Amino Acids↗
PubMed1982

Fluorescence characteristics of kynurenine and N'-formylkynurenine. Their use as reporters of the environment of tryptophan 62 in hen egg-white lysozyme.

Several kynurenine derivatives including N'-formylkynurenine were prepared in high purity by the ozonization of the corresponding indole compounds. The fluorescence characteristics of those derivatives were examined in connection with the use of their fluorophores as reporters for the local environment of tryptophan in proteins. Kynurenine is a weak emitter of fluorescence, with an emission maximum at 480 nm on excitation at 365 nm. With decreasing solvent polarity, the fluorescence intensity increases logarithmically and the emission maximum shifts to blue. A linear relation between these fluorescence characteristics and solvent polarity exists when the polarity is shown in terms of dielectric constant. N'-Formylkynurenine is a somewhat stronger emitter of fluorescence than kynurenine. The emission maximum is 434 nm on excitation at 325 nm and it shifts to blue in solvents of low polarity. This blue shift is also linear with respect to the dielectric constant of the solvent. Other factors influencing kynurenine fluorescence and N'-formylkynurenine fluorescence examined were neighboring groups, ionic strength, temperature, and protein denaturants. Based on the results of the present investigation, the local environment of tryptophan 62 in hen egg-white lysozyme was examined using Kyn 62-lysozyme.

Animals↗
PubMed1982

The role of the single tryptophan residue in the structure and function of ribonuclease T1.

The previously reported method for the preparation of Kyn 59-RNase T1 and NFK 59-RNase T1 has been improved, and these two proteins have been obtained in high purity. Kyn 59-RNase T1, fully active for the hydrolysis of GpA and GpC, emitted a 35-fold-enhanced fluorescence of kynurenine relative to acetylnurenine amide with an emission maximum at 455 nm upon excitation at 380 nm. The polarity of the environment of Kyn 59 estimated from the emission maximum corresponded to a dielectric constant of 6. Upon excitation at 325 nm, NFK 59-RNase T1, less active than Kyn 59-RNase T1, exhibited a quenched N'-formylkynurenine fluorescence with an emission maximum at 423 nm, from which the value of 12 was obtained as the dielectric constant of the surroundings of residue 59. In both modified proteins, distinct tyrosine fluorescence appeared on excitation at 280 nm. The detection of an energy transfer from tyrosine to residue 59 suggests that the tertiary structure is very similar in Kyn 59-RNase T1 and native RNase T1. With guanidine hydrochloride, Kyn 59-RNase T1 was less stable than the native protein. Carboxymethylation at Glu 58 was shown to stabilize the active site of the modified enzyme. Based on the information collected for Kyn 59-RNase T1, the local environment and possible roles of the sole tryptophan residue in RNase T1 are discussed.

Binding Sites↗
PubMed1982

Fluorescence titrations of residue 59 and tyrosine in Kyn 59-RNase T1 and NFK 59-RNase T1.

Fluorescence titrations of kynurenine and tyrosine in Kyn 59-RNase T1 and NFK 59-RNase T1 were carried out by monitoring protein fluorescence through a pH change from 1.5 to 10.5. In the titration of kynurenine fluorescence at 455 nm, a few small but distinct quenching events occurred between pH 3.5 and 9.5. Three ionizable groups were found to be responsible for the individual steps of quenching observed. These groups are Glu 58 with pKa 4.6, His 40 or 92 with pKa 7.8 and Lys 41 with pKa 8.7. From this result, a subtle conformational change associated with the proton dissociation equilibria of Glu 58 and His 40 or 92 in the active site of Kyn 59-RNase T1 is suggested. The pH-titration behavior of tyrosine fluorescence in Kyn 59-RNase T1 was different from that of kynurenine fluorescence. Two acidic groups with pKa's 3.2 and 6.5 were detected as perturbants. In NFK 59-RNase T1, both N'-formylkynurenine and tyrosine showed almost the same fluorescence behavior during titration, which was characterized by two transitions between pH 3 and 8 in each titration curve. Two ionizable groups with pKa's 3.7-3.8 and 6.7-6.8 were determined. The role of the latter ionizable group is discussed in relation to the enzyme function of RNase T1. From the close similarity in structure and function between Kyn 59-RNase T1 and RNase T1, it is suggested that the same mechanism of conformational change linked to the ionization states of Glu 58 and His 40 or 92 exists in the native protein too.

Hydrogen-Ion Concentration↗
PubMed1980

Binding of substrate analogs to hen lysozyme in which Trp 62 is modified to kynurenine.

The interactions of beta-methyl-GlcNAc, (GlcNAc)2, and (GlcNAc)3 with hen egg-white lysozyme [EC 3.2.1.17] in which Trp 62 is modified to kynurenine (Kyn 62-lysozyme) were studied by measuring the changes in the CD band and fluorescence due to the kynurenine at various pH values. The pH profiles of the binding constants of these saccharides to Kyn 62-lysozyme were very similar to those to intact lysozyme, although the binding constants were lower for the modified lysozyme than for intact lysozyme. The pK values of Asp 52, Glu 35, Asp 48, and Asp 101 in Kyn 62-lysozyme and in its complexes with beta-methyl-GlcNAc and with (GlcNAc)2 were in agreement with those of intact lysozyme and its complexes. The pK values of Asp 52 and Glu 35 in the modified lysozyme-(GlcNAc)3 complex were also in agreement with those of the complex with intact lysozyme, but the pK shift of Asp 101 was smaller for Kyn 62-lysozyme than for intact lysozyme. The significance of the decreased binding constants to Kyn 62-lysozyme is discussed. The pH dependence of the CD band due to the kynureinine in Kyn 62-lysozyme was interpreted in terms of the participation of the catalytic groups, Asp 52 (apparent pK 3.5) and Glu 35 (apparent pK 6.0), and the amino group of the kynurenine (apparent pK 0.75). This indicates that the ionization of the catalytic groups affects the state around Trp 62 and supports the previous proposal that there is a relation between the state around Trp 62 and the ionization of Glu 35 (Ikeda, K. & Hamaguchi, K. (1973) J. Biochem. 74, 221--230; (1975) ibid. 77, 1--16; Nakae et al. (1975) J. Biochem. 77, 993--1006). The intrinsic pK value of the amino group of the kynurenine at position 62 shifted from 2.1 to 0.8 on complexing with (GlcNAc)3, indicating between the kynurenine and the sugar residue at subsite B.

Animals↗
PubMed1979

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.

Amino Acids↗
PubMed1978

Chemical modification of ribonuclease T1 with ozone.

The single tryptophan residue in ribonuclease T1 [EC 3.1.4.8] was selectively oxidized by ozone to N'-formylkynurenine, which was then converted to kynurenine by acid-catalyzed deformylation in the frozen state. The two enzyme derivatives thus formed, NFK- and Kyn-RNase T1, lost enzymatic activity at pH 7.5, at which native RNase T1 most efficiently catalyzes the hydrolysis of RNA. At pH 4.75, the modified enzymes retained a decreased but distinct enzymatic activity toward RNA without alteration of substrate specificity, and Kyn-RNase T1 was four times more active than NFK-RNase T1. The binding of 3'-GMP to these modified enzymes decreased remarkably at pH 5.5, the optimum pH for binding to the intact enzyme. The gamma-carboxyl group of glutamic acid 58 was still reactive to iodoacetic acid after modification of tryptophan 59. The amounts of the carboxymethyl group introduced into NFK- and Kyn-RNase T1 were 0.36 and 0.59 mol, respectively, under conditions such that quantitative esterification of native RNase T1 takes place. CD spectroscopy indicated that the tertiary structure of the molecule was disordered in NFK-RNase T1, but not significantly in Kyn-RNase T1. It is concluded that tryptophan 59 functions in maintaining the active conformation of the protein structure, particularly in constructing the active environment for a functionally important set of groups involved in the binding of the substrate at the active site, although direct participation of in tryptophan the catalytic function of ribonuclease T1 is unlikely.

Chemical Phenomena↗
PubMed1978

Isolation of human urinary lysozyme.

For the isolation of human lysozyme from the urine of leukemia patients, a simple method has been established which involves precipitation of urinary proteins by 60% saturation with ammonium sulfate, fractionation of crude lysozyme on Sephadex G-50 and purification by CM-Sepharose chromatography. By this method approximately 60% of the lysozyme in the urine was isolated in a pure state in ten days.

Amino Acids↗
PubMed1976

Identification of tryptophan 62 as an ozonization-sensitive residue in hen egg-white lysozyme.

The position of a single N'-formylkynurenine residue in ozone-inactivated hen egg-white lysozyme [EC 3.2.1.17] was determined by two methods. One involved identification of an amino-terminus of the C-peptide obtained by selective cleavage with hydrazine of the kynurenyl peptide linkage in oxidized lysozyme. The other was to analyze a tryptic peptide containing kynurenine in the modified enzyme. Both methods showed that tryptophan 62 in lysozyme was so sensitive to ozone as to be selectively oxidized to N'-formylkynurenine with concomitant loss of the lytic activity.

Amino Acids↗
PubMed1976

Preparation and properties of trypsin-digested ribonuclease T1 split at the single arginyl peptide bond.

Ribonuclease T1 [EC 3.1.4.8] was selectively hydrolyzed by digestion with trypsin at the peptide bond of the single arginine residue at position 77. Selective hydrolysis was achieved by blocking the xi-amino group of Lys-41 with 2-methoxy-5-nitrotropone and subsequent digestion with trypsin in the presence of 2M urea. The trypsin-digested ribonuclease T1 was composed of two polypeptide chains containing 77 and 27 residues, though the two chains were covalently linked by a disulfide bond between Cys-6 and Cys-103. The modified enzyme lost enzymatic activity toward RNA and the ability to bind to 3'-GMP. The circular dichroic spectrum of the modified protein suggested that its conformation was extensively destroyed. It is concluded from the present results that the continuity of the peptide chain at the arginine residue is extremely important for maintaining the active conformation of the enzyme protein and for the enzymatic function of ribonuclease T1.

Amino Acids↗
PubMed1976

Enrichment of enzyme activity on deformylation of 1-NFK-lysozyme.

The formamide linkage of an inactive lysozyme derivative (1-NFK-lysozyme), formed by selective ozonization of tryptophan 62 in hen egg-white lysozyme [EC 3.2.1.17] was hydrolyzed with dilute acid faster in the frozen state at about --10 degrees than at 20 degrees. On hydrolysis of 1-NFK-lysozyme the low lytic activity increased to approximately 80% of that of native lysozyme. It is suggested that the binding ability associated with kynurenine 62 in the lysozyme derivative formed by this hydrolysis may be responsible for increase in enzymatic activity.

Binding Sites↗
PubMed1976

Preferential acid-catalyzed hydrolysis of the formamide linkage of N'-formylkynurenine in frozen solution.

Acid-catalyzed hydrolysis of the formamide linkage of N-acetyl-N'-formyl-L-kynurenineamide in frozen dilute hydrochloric acid solution followed first-order kinetics, yielding N-acetyl-L-kynurenineamide as the sole reaction product. The maximum rate of reaction in the frozen solution was found at around -7.5 degrees and approximated that of the reaction in liquid solution at 40 degrees. By freezing the dilute acid solution at -8 degrees the reaction was accelerated by 60 times compared with that in super-cooled liquid solution at the same temperature.

Formamides↗
PubMed1975

Oxidation of tryptophan in lysozyme by ozone in aqueous solution.

A tryptophan residue in hen's egg-white lysozyme [EC 3.2.1.17] was modified by ozone in an aqueous solution. One of the six tryptophan residues in the enzyme was oxidized to N'-formylkynurenine with concomitant loss of the enzymatic activity. Physicochemical studies of this modified enzyme (OL-I) revealed that the ozonization of lysozyme in aqueous media resulted in little change of the gross molecular conformation. It was deduced that the modified tryptophan residue in OL-I was possibly located in position 62 (or 63) of the protein.

Amino Acids↗