A comparison of the effects of ultraviolet and ionizing radiations on trypsin activity and on its constituent amino acids

Photons of 254nm. u.v. light, (60)Co gamma-rays and 1Mev electrons produce different patterns of destruction of individual amino acids in dried films of trypsin and in the corresponding amino acid mixture. For example, in the amino acid mixture u.v. light destroys tyrosine, tryptophan and cystine, whereas in trypsin only cystine is disrupted but with 10 times the initial yield. Further, in the amino acid mixture loss of half-cystine is a simple exponential function of dose, but in trypsin there appear to be two exponential components of the loss with yields that differ by a factor of 35. Both the gamma-rays and electrons destroy half-cystine, tryptophan, histidine and methionine in the amino acid mixture with remarkably high yields, whereas in trypsin doses that destroy almost all of the enzymic activity produce no detectable destruction of amino acid residues. These marked differences between the two preparations show that the radiation-sensitivity of a given amino acid alone and in a protein is different, and suggests that in trypsin there is fairly extensive migration of energy, charge or both with localization of damage at specific sites determined by this enzyme's internal organization. All three types of radiation produce appreciable amounts of ;damaged' (not completely inactivated) molecules which are prevented from reassuming an active configuration by the addition of 5.5m-urea; thiol reagents have a similar effect after bombardment with u.v. light or electrons. The patterns of destruction produced by gamma-rays and by electrons in both the amino acid mixture and in trypsin are different (some of the yields vary by a factor of 30). This result appears to be inconsistent with the popular belief that most of the energy absorbed from gamma-rays is associated with very-high-energy electrons.     

Ribonuclease of Bacillus intermedius 7 P. Purification by chromatography on phosphocellulose and several characteristics of the homogeneous enzyme]

A new procedure for isolation of homogenous ribonuclease of Bac. intermedius from a commercial source is described. The yields of 140 mg of RNAse from 200 g of the enzymic powder were attained. The amino acid composition of the enzyme was determined. The RNAse contains neither the sulfhydryl groups nor the disulfide bonds and has only one histidine residue. At the same time the amount of aromatic amino acid residues is relatively high. The enzyme is highly resistant to heat and acid treatment but is less stable in an alkaline solution. The pH optimum of the RNAse for the RNA digestion is 8,5; the temperature optimum for this reaction is 37 degrees. A spectrophotometric method for the RNAse activity assay using polyA as a specific substrate was developed. The purified product provides a suitable starting material for structural studies.     

Immunochemical reactivity of native and modified preparations of pig kidney diamine oxidase

This paper describes the antigenicity of pig kidney diamine oxidase [EC 1.4.3.6] and the possible role of constituent amino acids in the epitope structure of the enzyme. The loss of 62% of the biological activity after DAO-anti-DAO antibodies interaction was attributed to the steric hindrance caused by binding of antibody to the enzyme molecule. A gradual loss in antigenicity during ultraviolet (UV) irradiation was observed without any significant conformational change, demonstrating the destruction of antigenic determinants. However, ethoxyformylation of nine histidyl residues with complete inactivation caused no change in immunoreactivity. The results indicate that the antigenic sites and catalytic sites are located at different positions along the polypeptide chain. Moreover, the results of lysine residue modification were suggestive of possible involvement of lysine in the antigenic determinants of DAO.     

Aspartokinase-homoserine dehydrogenase I from Escherichia coli: pH and chemical modification studies of the kinase activity

The pH variation of the kinetic parameters was examined for the kinase activity of the bifunctional enzyme aspartokinase--homoserine dehydrogenase I isolated from Escherichia coli. The V/K profile for L-aspartic acid indicates the loss of activity upon protonation of a cationic acid type group with a pK value near neutrality. Incubation of the enzyme with diethyl pyrocarbonate at pH 6.0 results in a loss of enzymic activity. The reversal of this reaction by neutral hydroxylamine, the appearance of a peak at 242 nm for the inactivated enzyme, and the observation of a pK value of 7.0 obtained from variation of the inactivation rate with pH all suggest that enzyme inactivation occurs by modification of histidine residues. The substrate L-aspartic acid protects one residue against inactivation, which implies that this histidine may participate in substrate binding or catalysis. Activity loss was also observed at high pH due to the ionization of a neutral acid group with a pK value of 9.8. The reactions of AK-HSD I with N-acetylimidazole and tetranitromethane have been investigated to obtain information about the functional role of tyrosyl residues in the enzyme. The acylation of tyrosines leads to inactivation of the enzyme, which can then be fully reversed by treatment with hydroxylamine. Incubation of the enzyme with tetranitromethane at pH 9.5 also leads to rapid inactivation, and the substrates of the kinase reaction provide substantial protection against inactivation. However, three tyrosines are protected by substrates, implying a structural role for these amino acids.     

Modification of uridine phosphorylase from Escherichia coli by diethyl pyrocarbonate. Evidence for a histidine residue in the active site of the enzyme.

Uridine phosphorylase from Escherichia coli is inactivated by diethyl pyrocarbonate at pH 7.1 and 10 degrees C with a second-order rate constant of 840 M-1.min-1. The rate of inactivation increases with pH, suggesting participation of an amino acid residue with pK 6.6. Hydroxylamine added to the inactivated enzyme restores the activity. Three histidine residues per enzyme subunit are modified by diethyl pyrocarbonate. Kinetic and statistical analyses of the residual enzymic activity, as well as the number of modified histidine residues, indicate that, among the three modifiable residues, only one is essential for enzyme activity. The reactivity of this histidine residue exceeded 10-fold the reactivity of the other two residues. Uridine, though at high concentration, protects the enzyme against inactivation and the very reactive histidine residue against modification. Thus it may be concluded that uridine phosphorylase contains only one histidine residue in each of its six subunits that is essential for enzyme activity.     

Characteristics of the effect of gamma-irradiation on the amino acid composition of collagen as modified by a gaseous atmosphere

The comparative changes in the amino acid composition of calf skin collagen after gamma-irradiation (doses from 100 to 1,000 Gy) in aqueous solutions under different gas atmospheres (O2, N2O, H2, vacuum) were investigated. The radiochemical yields of collagen amino acid residues destruction were determined. Under O2 (OH X, O2-) most of amino acids are destroyed with higher yields than under N2O. Leucine, valine, isoleucine, phenylalanine, arginine were the exception because of their high reaction rate constants with OH X and hydroxylation reactions. Under H2 (e-aq, H) and in vacuum (e-aq, OH X) the mechanism of collagen radiolysis changed due to its aggregation; the destruction of those amino acids which have high reaction rate constants with water radiolysis products was mainly observed (phenylalanine, tyrosine, histidine).     

Photo-oxidation and carboxymethylation of guanylribonuclease Pch1]

The effect of photo-oxidation and carboxymethylation on the activity of RNAse Pch1 has been studied. Photoinactivation in the presence of rose bengal results in a selective oxidation of two histidine residues. The process is inhibited by the nucleotide substrate analogs. This suggests that one or two imidazole groups may be localized in the active site of RNAse Pch1. The pH dependence of the enzyme inactivation by bromoacetic acid is indicative of the contribution of a functional group with pKa 4,0, presumably of a beta- or gamma-carboxyl group of dicarbonic amino acid. The reaction is inhibited by the substrate analogs 2'(3')-GMP and 2'(3')-AMP. The data on the similarity of active sites in several guanyloribonucleases are discussed.     

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.     

Ein Vergleich der Wirkung von ionisierender Strahlung auf peptidgebundene und freie Aminosäuren in festem Zustand

Zusammenfassung Untersuchungen an thermischen Polymeren von-Aminosäuren in festem Zustand zeigen, daß in diesen insbesondere Tryptophan, Histidin, Cystin, Lysin und Methionin eine höhere Strahlenempfindlichkeit als in den bisher untersuchten Proteinen aufweisen. Diese Ergebnisse werden verglichen mit ähnlichen Untersuchungen an Filmen von Aminosäuremischungen, die in noch stärkerem Umfang auf einen beträchtlichen Energietransfer oder Chargetransfer in Richtung auf die vier genannten Aminosäuren schließen lassen. Die Ergebnisse werden auch in Hinsicht auf die Strahlenempfindlichkeit von Aminosäuren in Proteinen und auf die Inaktivierung von Enzymen diskutiert.

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A comparison of the effect of ionizing radiation on peptide- bound and free amino acids in the solid state

Summary Irradiation of thermal polymers of-amino acids with X-rays in the solid state produces a significantly increased destruction of tryptophan, histidine, cystine, lysine and methionine as compared with the response of constituent amino acids in proteins. These results are discussed with respect to related results obtained by irradiation of dry films of amino acid mixtures which indicate an even stronger energy or charge transfer towards the four amino acids mentioned. The results are also discussed with respect to the radiation sensitivity of constituent amino acids in proteins and the inactivation of enzymes.

     

The primary structure of crotamine (author's transl)]

The primary structure of crotamine, a basic toxin isolated from the venom of the South American rattle-snake Crotalus durissus terrificus has been determined. The polypeptide chain is composed of 42 residues of amino acids. Crotamine shows a molecular weight of 4900 and contains 6 half cystine, 9 lysine, 2 arginine, 2 histidine and 2 tryptophan residues.     

The involvement of one of the three histidine residues of cow kappa-casein in the chymosin-initiated milk clotting process.

Cow kappa-casein has been modified by photo-oxidation in the presence of rose bengal and by the chemical reagents diethyl pyrocarbonate, 2-hydroxy-5-nitro-benzyl bromide and iodoacetic acid. Photo-oxidation resulted in the destruction of histidine and tryptophan residues and all of the histidines could be ethoxy-formylated by treatment with diethyl pyrocarbonate. Both procedures caused a loss in the susceptibility of the Phe-Met linkage of kappa-casein to chymosin hydrolysis. Treatment of kappa-casein with 2-hydroxy-5-nitrobenzyl bromide and iodoacetic acid caused the loss of tryptophan and methionine residues respectively but, in both cases, the susceptibility of the modified protein to chymosin hydrolysis remained unaffected. Of the amino acids examined it is concluded that only the histidine residues of cow kappa-casein are important for the hydrolytic action of chymosin and, furthermore, the treatment with diethyl pyrocarbonate suggests that only one of the three histidines plays an essential role.     

Chemical modification of cholesterol-hydroxylating cytochrome P-450 from adrenal cortex mitochondria with diethylpyrocarbonate

Chemical modification of adrenocortical cytochrome P-450scc with diethyl pyrocarbonate has been carried out. The histidine residues and the protein amino groups were shown to undergo modification. Carbethoxylation was accompanied by the hemoprotein inactivation and the loss of enzymatic activity. Neither of the high spin effectors (i.e., substrate and adrenodoxin) protected cytochrome P-450scc either from inactivation or from the loss of enzymatic activity. The data obtained are discussed in terms of the functional role of histidine residues in the cytochrome P-450scc molecule.     

The presence of essential histidine residues in manganese(III)-containing acid phosphatase from sweet potato

Chemical modification studies of manganese(III)-containing acid phosphatase [EC 3.1.3.2] were carried out to investigate the contributions of specific amino-acid side-chains to the catalytic activity. Incubation of the enzyme with N-ethylmaleimide at pH 7.0 caused a significant loss of the enzyme activity. The inactivation followed pseudo-first-order kinetics. Double log plots of pseudo-first-order rate constant vs. concentration gave a straight line with a slope of 1.02, suggesting that the reaction of one molecule of reagent per active site is associated with activity loss. The enzyme was protected from inactivation by the presence of molybdate or phosphate ions. Amino acid analyses of the N-ethylmaleimide-modified enzyme showed that the 96%-inactivated enzyme had lost about one histidine and one-half lysine residue per enzyme subunit without any significant decrease in other amino acids, and also demonstrated that loss of catalytic activity occurred in parallel with the loss of histidine residue rather than that of lysine residue. Molybdate ions also protected the enzyme against modification of the histidine residue. The enzyme was inactivated by photooxidation mediated by methylene blue according to pseudo-first-order kinetics. The pH profile of the inactivation rates of the enzyme showed that an amino acid residue having a pKa value of approximately 7.2 was involved in the inactivation. These studies indicate that at least one histidine residue per enzyme subunit participates in the catalytic function of Mn(III)-acid phosphatase.     

Variation in the free amino acid content of skeletal muscle of Ophicephalus punctatus (Bloch)

The skeletal muscle of Ophicephalus punctatus contains nine essential free amino acids, arginine, histidine, isoleucine, leucine, methionine, phenylalanine, threonine, valine and lysine, and eight non-essential amino acids, alanine, aspartic acid, cystine, glutamic acid, glycine, tyrosine, proline and serine. Histidine and lysine dominated the free amino acids pool. Seasonal variation was detected in the levels of histidine, arginine, leucine, phenylalanine, glycine, cystine and serine with highest values occurring in April and again in November. Changes were also detected in the concentrations of certain amino acids as the fish grew in size. Levels of free amino acids did not significantly differ between sexes. Factors effecting variation are discussed.     

Chemical modification of A1 adenosine receptors in rat brain membranes. Evidence for histidine in different domains of the ligand binding site

Chemical modification of amino acid residues was used to probe the ligand recognition site of A1 adenosine receptors from rat brain membranes. The effect of treatment with group-specific reagents on agonist and antagonist radioligand binding was investigated. The histidine-specific reagent diethylpyrocarbonate (DEP) induced a loss of binding of the agonist R-N6-[3H] phenylisopropyladenosine ([3H]PIA), which could be prevented in part by agonists, but not by antagonists. DEP treatment induced also a loss of binding of the antagonist [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX). Antagonists protected A1 receptors from this inactivation while agonists did not. This result provided evidence for the existence of at least 2 different histidine residues involved in ligand binding. Consistent with a modification of the binding site, DEP did not alter the affinity of [3H]DPCPX, but reduced receptor number. From the selective protection of [3H] PIA and [3H]DPCPX binding from inactivation, it is concluded that agonists and antagonists occupy different domains at the binding site. Sulfhydryl modifying reagents did not influence antagonist binding, but inhibited agonist binding. This effect is explained by modification of the inhibitory guanine nucleotide binding protein. Pyridoxal 5-phosphate inactivated both [3H]PIA and [3H]DPCPX binding, but the receptors could not be protected from inactivation by ligands. Therefore, no amino group seems to be located at the ligand binding site. In addition, it was shown that no further amino acids with polar side chains are present. The absence of hydrophilic amino acids from the recognition site of the receptor apart from histidine suggests an explanation for the lack of hydrophilic ligands with high affinity for A1 receptors.     

Inactivation of 3 alpha-hydroxysteroid dehydrogenase by superoxide radicals. Modification of histidine and cysteine residues causes the conformational change.

3 alpha-Hydroxysteroid dehydrogenase (EC 1.1.1.50) from Pseudomonas testosterone was inactivated by superoxide radicals generated by the aerobic xanthine oxidase reaction. Superoxide dismutase, NAD+, bovine serum albumin and histidine and cysteine as free amino acids partially protected the enzyme from inactivation. NADH-binding properties were determined by fluorescence spectroscopy, and no variation was found between native enzyme and the unmodified fraction of the partly inactivated one. The fluorescence emission maximum for the completely inactivated enzyme was shifted 10 nm to a longer wavelength when compared with the native one, and it seems possible that the modification of histidine and cysteine residues by superoxide radicals causes the conformational change of the enzyme and the consequent loss of catalytic activity.     

Photochemical oxidation of snake gourd proteinase A2, a serine proteinase

Snake gourd proteinase A₂ was rapidly inactivated by methylene blue catalysed photooxidation at pH 7.8 and 25°. The rate of inactivation was pH-dependent and became slower at lower pH values, suggesting the involvement of some histidine residues in the inactivation. Changes in amino acid composition occurred only with histidine residues. One mole or more of histidine residues in the molecule are of essential importance in the catalytic function of snake gourd proteinase A₂.     

Features of a Spatially Constrained Cystine Loop in the p10 FAST Protein Ectodomain Define a New Class of Viral Fusion Peptides

The reovirus fusion-associated small transmembrane (FAST) proteins are the smallest known viral membrane fusion proteins. With ectodomains of only ∼20–40 residues, it is unclear how such diminutive fusion proteins can mediate cell-cell fusion and syncytium formation. Contained within the 40-residue ectodomain of the p10 FAST protein resides an 11-residue sequence of moderately apolar residues, termed the hydrophobic patch (HP). Previous studies indicate the p10 HP shares operational features with the fusion peptide motifs found within the enveloped virus membrane fusion proteins. Using biotinylation assays, we now report that two highly conserved cysteine residues flanking the p10 HP form an essential intramolecular disulfide bond to create a cystine loop. Mutagenic analyses revealed that both formation of the cystine loop and p10 membrane fusion activity are highly sensitive to changes in the size and spatial arrangement of amino acids within the loop. The p10 cystine loop may therefore function as a cystine noose, where fusion peptide activity is dependent on structural constraints within the noose that force solvent exposure of key hydrophobic residues. Moreover, inhibitors of cell surface thioreductase activity indicate that disruption of the disulfide bridge is important for p10-mediated membrane fusion. This is the first example of a viral fusion peptide composed of a small, spatially constrained cystine loop whose function is dependent on altered loop formation, and it suggests the p10 cystine loop represents a new class of viral fusion peptides.     

Conversion of amino acid residues in proteins and amino acid homopolymers to carbonyl derivatives by metal-catalyzed oxidation reactions

A number of metal-catalyzed oxidation (MCO) systems mediate the oxidative inactivation of enzymes. This oxidation is accompanied by conversion of the side chains of some amino acid residues to carbonyl derivatives (for review, see Stadtman, E. R. (1986) Trends Biochem. Sci. 11, 11-12). To identify the amino acid residues which are sensitive to MCO oxidation, several enzymes/proteins and amino acid homopolymers were exposed to various MCO systems. The carbonyl groups which were formed were converted to their corresponding 3H-labeled hydroxy derivatives. After acid hydrolysis, the labeled free amino acids were separated by ion exchange chromatography. Each protein or polymer gave rise to several different labeled amino acids. The elution profiles of the labeled amino acids obtained from preparations of Escherichia coli glutamine synthetase which had been oxidized by MCO systems comprised of either Fe(II)/O2 or ascorbate/Fe(II)/O2 both in the presence and absence of EDTA were qualitatively the same. From a comparison of the elution profiles of labeled amino acids from various proteins with those obtained from homopolymers, it is evident that the side chains of histidine, arginine, lysine, and proline are particularly sensitive to oxidation by the MCO systems. This conclusion is supported also by direct amino acid analysis of acid hydrolysates which shows that the oxidation of glutamine synthetase, enolase, and phosphoglycerate kinase is associated with the loss of at least 1 histidine residue per subunit. From the results of studies with homopolymers, it is apparent that glutamic semialdehyde is a major product of both proline and arginine residues. In addition, hydroxyproline and unlabeled glutamic acid were identified among the hydrolysis products of oxidized poly-L-proline, and unlabeled aspartic acid was identified as a product of poly-L-histidine oxidation.     

Mutational analysis of histidine residues in the human proton-coupled amino acid transporter PAT1

The proton-coupled amino acid transporter 1 (PAT1) represents a major route by which small neutral amino acids are absorbed after intestinal protein digestion. The system also serves as a novel route for oral drug delivery. Having shown that H+ affects affinity constants but not maximal velocity of transport, we investigated which histidine residues are obligatory for PAT1 function. Three histidine residues are conserved among the H+-coupled amino acid transporters PAT1 to 4 from different animal species. We individually mutated each of these histidine residues and compared the catalytic function of the mutants with that of the wild type transporter after expression in HRPE cells. His-55 was found to be essential for the catalytic activity of hPAT1 because the corresponding mutants H55A, H55N and H55E had no detectable l-proline transport activity. His-93 and His-135 are less important for transport function since H93N and H135N mutations did not impair transport function. The loss of transport function of His-55 mutants was not due to alterations in protein expression as shown both by cell surface biotinylation immunoblot analyses and by confocal microscopy. We conclude that His-55 might be responsible for binding and translocation of H+ in the course of cellular amino acid uptake by PAT1.