Investigation of the active sites of lysozyme. Carboxymethylation of the imidazole group of histidine and of the ϵ-aminogroup of lysine

Кравченко, Н. А.; Kleopina, G. V.; Kaverzneva, E. D.

doi:10.1016/0926-6569(64)90205-6

Cited by 8 publications

(10 citation statements)

References 9 publications

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“…Coulombic interactions have been investigated by increasing the ionic strength of the buffer or by reducing the positive charge on the protein [8,9]. However, the effects of adding an extra negative charge to the protein has not previously been considered.…”

Section: Electrostatic Factors Affecting Cell-wall Hydrolysismentioning

confidence: 99%

“…Previous studies on the hydrolysis of M. luteus cell walls have implicated the electrostatic interaction between basic groups on the surface of lysozyme and the cell wall as the dominant event. Evidence for this has come from two sources : firstly, the pHdependence of cell-wall hydrolysis shows a pK a of 9-10 associated with ε-NH # groups [5] ; and secondly, acetylation or methylation of lysine residues produces a species that has reduced, or no lyticactivity against M. luteus [8,9]. The mechanism of lysis, as demonstrated on neutral substrates (such as oligomers of chitin), is not affected by acetylation or methylation of lysine residues, rather the effect is on the interaction with the cell-wall substrate [9].…”

Section: Effects Of Nitration On Cell-wall Hydrolysismentioning

confidence: 99%

“…It has long been known that the high pI of lysozyme is important for its ability to hydrolyse rapidly the negatively charged cell walls of Gram-positive bacteria ; this is a defining feature of this family of proteins [7]. Previous studies into this property have relied on chemical modification of lysine and arginine residues to reduce the surface charge of the protein [8,9]. These experiments revealed a decrease in cell wall lytic activity, dependent upon ionic strength, with little or no loss in activity against uncharged substrates nor any structural changes within the protein [10].…”

Section: Introductionmentioning

confidence: 99%

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The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme

Richards

Walton

Heptinstall

1996

Biochemical Journal

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We have investigated the effects of tyrosine nitration (to form the weak acid, 3-nitrotyrosine) at positions 23 or 20 plus 23, on the structure and function of hen egg-white lysozyme. Enzyme activity against Micrococcus luteus cell-wall fragments or soluble substrates exhibits two phenomena. (a) A decrease in Km and kcat for the hydrolysis of soluble oligo- and poly-saccharides, resulting in only minor changes in the catalytic efficiency (kcat/Km) upon nitration. (b) The hydrolysis of M. luteus cell-wall fragments appeared to be dominated by electrostatic interactions with the protein, giving a decrease in enzyme activity as the 3-nitrotyrosyl group became ionized. Removal of the cell-wall anionic polymer, teichuronic acid, from M. luteus abolished this effect. The 3-nitrotyrosine group was also found to act as a fluorescence quencher of exposed tryptophan residues in lysozyme.

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Section: Electrostatic Factors Affecting Cell-wall Hydrolysismentioning

confidence: 99%

Section: Effects Of Nitration On Cell-wall Hydrolysismentioning

confidence: 99%

Section: Introductionmentioning

confidence: 99%

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The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme

Richards

Walton

Heptinstall

1996

Biochemical Journal

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“…Nevertheless, this residue iodinates and alkalytes readily [8,36]. It is tempting to postulate that histidine 16 reactivity may be aided by electrostatic facilitation from the guanidinium group of arginine 14.…”

Section: Conformational Effectsmentioning

confidence: 99%

Factors in the Iodination of Histidine in Proteins

Wolff

Covelli

1969

European Journal of Biochemistry

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Factors that determine the reactivity toward iodine of the histidyl residues of various proteins are compared. The proteins studied include lysozyme, ribonuclease, insulin, lactic, malic and glutamic dehydrogenases, phosphoglucomutase, thyroglobulin, trypsin, chymotrypsin and #?-lactoglobulin. Relatively fewer histidyl residues than tyrosyl residues are iodianated a t pH 8.5 both in water and in guanidine. The fraction of iodine present in histidyl residues increases with increasing pH. Diiodohistidine is formed primarily in proteins that do not contain tryptophane.Thyroglobulin can be iodinated to contain So/, histidine iodine. However, mono-and diiodohist,idine formation in equilibrium-labelled thyroids accounts for < lo/, of the total iodine.Histidine iodination requires the dissociable proton of the imidazole ring as shown by lack of iodination of N-methylated model compounds. The assumption that iodination proceeds by an attack on the imidazole anion (rather than via an N-I intermediate) is supported by the finding that the sterically hindered 2-t-butyl imidazole is iodinated.It is concluded that, although conformational factors such as electrostatic facilitation and ligand binding influence the reactivity of certain histidyl residues, the major factors in histidyl iodination are the instrinsic properties of the residue.Indirect evidence for the iodination of histidyl residues in proteins was adduced long ago by Bauer and Strauss [l] for globin and by Fraenkel-Conrat [2] for lysozyme. More recently, the decrease of histidyl residues in hydrolysates of iodoproteins has been taken as evidence that iodohistidyl residues had been formed 13-61, and it is generally held that iodohistidines are destroyed during acid hydrolysis [2,3,5,6]. It is well established, although not generally appreciated, that mono-and diiodohistidyl residues are readily formed during the iodination of a variety of proteins in vitro. Roche et al. [7] demonstrated the presence of iodohistidines in globin and thyroglobulin. With quantitative chromatographic techniques it has been possible to show that monoiodohistidine and/or diiodohistidine are formed in good yield during the iodination of lysozyme [S], pancreatic ribonuclease A [9], fructose 1,6-diphosphate [lo], and insulin [ll]. I n lysozyme and insulin up to one third of the total organic iodine was present in histidyl residues.There was considerable variation in the reactivity of different histidyl residues of the same protein. This was influenced, in part, by the presence of certain hgands believed to bind to histidine [9,11], and presumably in part by conformational factors. We therefore attempted t o assess the importance of histidine iodination in a series of other proteins, and in particular, thyroglobulin; moreover, it was important to decide, if possible, whether incomplete iodination was due primarily to increased reactivity of some residues or low reactivity of the others. We also studied certain aspects of histidine iodination with the hope of differentiating intrinsic react...

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“…Increase in activity of CM-His lysozyme was also obtained by Kravchenko et al [19]. Moreover, when we plotted the evolution of the enzymatic activity as a function of carbamylation a t pH 8.5 (Fig.2) it appeared that carbamylation of one lysine seemed to increase lysozyme activity.…”

Section: Enzymatic Activity Of the Djj?ferent Lysozyme Derivativesmentioning

confidence: 77%

Immunochemical Studies on Hen's Egg‐White Lysozyme

Strosberg¹,

Kanarek²

1970

European Journal of Biochemistry

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By specific chemical modification of amino acid residues of egg white lysozyme it is demonstrated that the carbamylation of the cc amino group and carboxymethylation of the unique histidine residue do not affect the reaction of the enzyme with the anti-lysozyme antibodies.I n contrast, it appears from this work that carbamylation or acetylation of the E amino groups, carboxymethylation of the methionine residues as well as CNBr cleavage of the polypeptide chain do alterate the antigenic structure of lysozyme.Thanks to the work of Jollks and coworkers [l], of Canfield [Z], and of Blake et al.[3] the sequence and the spatial conformation of hen's egg white lysozyme are well determined. Therefore, this enzyme appears as an ideal choice to study the antigenic determinants of a globular protein. The characterization of Werent amino acid residues implicated in the antigenic sites will not only allow these sites to be localized but will also assist in obtaining some information on their spatial organization.Different approaches can be used for the determination of the antigenic sites of a protein. One of these approaches consists in the cleavage of the protein into large fragments by enzymatic or chemical methods and in the characterization of the fragments that retain some combining activity with antibodies directed to the native antigen. Fujio et al. have succeeded in isolating pepsic fragments of lysozyme still containing antigenic determinants of the native enzyme. This type of approach will, however, not allow, the identification of all the sites made up by residues widely separated in the unfolded chain, but in close proximity through the speci6c folding of the protein.I n the case of lysozyme, this type of "conformational determinant" seems very important, as reduced and amino-ethylated enzyme is unable to combine with any of the rabbit or goat anti-lysozyme antibodies which have been tested. This observation is comparable with the observation made by Brown et al.[8] in the case of oxidized ribonuclease. This similarity in behaviour of lysozyme and ribonuclease is probably due to the importance of the disulfide bridges in maintaining the conformation of these proteins.We have therefore chosen to study the influence of chemical modifications of certain residues on the immunological activity of lysozyme. Different attempts have been made to identify the lysozyme residues involved in the interaction lysozyme-antilysozyme antibodies. Fujio et al. I n this paper we report the effect on the lysozyme anti-lysozyme antibody interaction of carbamylation of the free amino groups, carboxymethylation of the single histidine and of the methionine residues and we confirm the results obtained by Bonavida, Miller and Sercarz [17] by cleavage of the polypeptide chain a t the methionine residues of the enzyme. MATERIALS AND METHODSThree times recrystallized egg-white lysozyme was purchased from Sigma Chemical (St. Louis, Missouri) lot no 127B-8030 and was used without further purification. When chromatographed on a carboxylic ...

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Investigation of the active sites of lysozyme. Carboxymethylation of the imidazole group of histidine and of the ϵ-aminogroup of lysine

Cited by 8 publications

References 9 publications

The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme

The effects of tyrosine nitration on the structure and function of hen egg-white lysozyme

Factors in the Iodination of Histidine in Proteins

Immunochemical Studies on Hen's Egg‐White Lysozyme

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