Lysyl group modification

Measurement of Lysyl Group Modification. The concentration of lysyl E-amino groups in collagen was determined by the method of Porter et al.(20). A fifteen mg sample of air dried collagen... 

Figure 3. Sorption of fS-galactosidase fE. coli Km) by collagen preparations at different levels of lysyl group modification...

The reactivity of the lysyl residues in NADH-cytochrome 65 reductase has been investigated and found to fall into three groups. Modification of the first most reactive group (about half the total) results in loss of the ability to interact with cytochrome bg. In the presence of NADH all but one of the remaining lysyl residues react, resulting in destabilization of the holoenzyme structure (349). 

Figure 1 illustrates the side chains of proteins useful for the formation of neoglycoproteins. Among them, the e-amino group of lysyl side chains is the most frequently used for modification of proteins, because the lysyl group is often abundant, and its side chain is flexible and solvent accessible. In the next four sections, we will describe the sugar attachment via these four functional groups. 

A final cardinal feature of IsoK/LG biochemistry is their propensity to disrupt protein function. Modification of proteins by IsoK/LG could theoretically alter protein function by several mechanisms. Adduction of IsoK/LG to lysine converts a short, positively charged group to a bulky, hydrophobic, negatively charged group. Modifications of lysyl residues in the active site of enzymes will therefore eliminate catalytic activity. Catalytic activity may be lost even if the modified residue is simply adjacent to the active site, as the bulky IsoK/LG adduct may sterically hinder substrate binding or product release. Protein... 

Tropoelastin molecules are crosslinked in the extracellular space through the action of the copper-dependent amine oxidase, lysyl oxidase. Specific members of the lysyl oxidase-like family of enzymes are implicated in this process (Liu etal, 2004 Noblesse etal, 2004), although their direct roles are yet to be demonstrated enzymatically. Lysyl oxidase catalyzes the oxidative deamination of e-amino groups on lysine residues (Kagan and Sullivan, 1982) within tropoelastin to form the o-aminoadipic-6-semialdehyde, allysine (Kagan and Cai, 1995). The oxidation of lysine residues by lysyl oxidase is the only known posttranslational modification of tropoelastin. Allysine is the reactive precursor to a variety of inter- and intramolecular crosslinks found in elastin. These crosslinks are formed by nonenzymatic, spontaneous condensation of allysine with another allysine or unmodified lysyl residues. Crosslinking is essential for the structural integrity and function of elastin. Various crosslink types include the bifunctional crosslinks allysine-aldol and lysinonorleucine, the trifunctional crosslink merodes-mosine, and the tetrafunctional crosslinks desmosine and isodesmosine (Umeda etal, 2001). 

Figure 1. The effect of chemical modification of lysyl E-amino groups on the sorption of fi-galacatosidase (23 pmol/L) to collagen membrane. Solution equilibrium and steady-state reactor conditions.

Topaquinone and lysyltopaquinone are formed by postsynthetic modification of the precursor proteins of the active enzymes. A tyrosine residue in the enzyme undergoes autocatalytic oxidation in the presence of enzyme-bound copper and oxygen. Lysyltopaquinone in lysyl oxidase is believed to be synthesized by reaction between topaquinone and the e-amino group of a lysine residue to form the cross-linked imino adduct. This means that it is highly improbable that either topaquinone or lysyltopaquinone is a dietary essential, because there is no way in which preformed quinone could be incorporated into the precursor protein or a hypothetical apoenzyme. 

In the development of proteins as therapeutic agents, it is crucial to ensure the homogeneity of the potential products. However, a survey of the current literature has indicated that overexpression of recombinant proteins in E. coli often leads to translational errors as well as post-translational modifications (1). Acetylation of the e-amino group of lysyl residues in bovine somatotropin has been documented (2). Substitution of methionine by norleucine is another well known example (3,4). 

The e-amino groups of lysyl residues in proteins may be carbamylated by cyanate to give homocitrulline residues ( 3.1.2.1). It should be noted that some carbamylation of proteins can occur in urea solutions that have not been deionized to remove cyanate. The extent of modification is often difficult to assess, however, since acid hydrolysis of fully carbamylated proteins under the usual conditions gives homocitrulline plus 17-33% free lysine (Stark and Smyth 1963). Although the lysine recovery (in %) is variable, depending on the protein studied, for a given protein the lysine recovery is apparently constant. 

The -amino groups of lysyl residues serve as attachment sites of a number of coenzymes in proteins (e.g. biotin in pyruvate carboxylase, pyridoxal phosphate in phosphorylase, lipoic acid in lipoate acetyl-transferase) and form covalent intermediates in several enzymic reactions (e.g. transaldolase, aldolase, etc.). Discussion of all of these naturally-occuring derivatives of lysine will not be attempted in this treatise, but the investigator using chemical modification of proteins should be aware of their possible presence and effect on the results of his experiments. It should be noted that e-N-phospholysine has been reported in nucleoside diphosphate kinase (Walinder 1968). 

Studying chemical modification will be described. Histones are modified in vivo by acetylation (also methylation and phosphorylation, see DeLange and Smith 1971, 1974). In some histones the a-amino groups are acetylated (in addition to lysine residues) and this precludes direct examination of the internal sites of acetylation by the Edman procedure. However, histones IIb2 and III have free a-amino groups and this enabled Candido and Dixon (1972) to examine these histones (from trout testis) which had been acetylated intracellularly with " C-labeled acetate. Each residue that was released by the Edman procedure (using a sequenator) was examined for radioactive material and it was found that lysyl Residues 14 and 23 (major sites) and 9 and 18 (minor sites) were partially acetylated in histone III and lysyl Residues 5,10,13 and 18 were partially acetylated in histone IIb2. This type of approach should be applicable to many other studies of chemical modification. 

Lysine tyrosylquinone (LTQ) (Figure 3) is the protein-derived cofactor of mammalian lysyl oxidase, an important enzyme in the metabolism of connective tissue. Lysyl oxidase catalyzes the posttranslational modification of elastin and collagen. It oxidizes selected peptidyl lysine residues to peptidyl a-aminoadipic -semialdehyde residues. This initiates formation of the covalent cross-linkages that insolubilize these extracellular proteins. This enzyme also contains copper as a second prosthetic group. 

Analysis of acid hydrolyzates of the substrate-protected carboi lase and comparison of the elution position of tritium from the amino acid analyzer with standards prepared from glutathione, whose —SH group had been modified with Br-butanone-P2, showed that under protective conditions only —SH groups of the carboxylase were modified. In contrast, identical experiments with the inactivated carboxylase coupled with characterization of tryptic peptides showed that lysyl residues, in addition to cysteinyl residues, were modified. Thus, inactivation is a consequence of modification of lysyl residues. 

SO that the difference between tritium incorporation in the inactivated and protected samples is not a true reflection of the number of residues protected by ribulosebisphosphate from modification. However, in the inactivated sample the difference between the incorporation based on tritium (13.8 moles of reagent per mole of enzyme) and the number of —SH groups modified (8) should represent the number of lysyl residues (5.8) that react with Br-butanone-Pa during inactivation. The extent of incorporation based on is low because of the instability of the phosphate group a to the carbonyl. ... 

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