Carbamylation of lysine

Note Incubation at higher temperature may cause urea-based carbamylation of lysines and protein N-termini. 

When cyanate is administered to animals, most of the radioactivity is accounted for by carbamylation of NH2-terminal residues of hemoglobin. No detectable carbamylation of lysine or cystine residues is observed. The exact mechanism by which cyanate protects against sickling is not known. Because cyanate... 

Resuspend the dried protein in 50 yuL of fresh 8 M urea-80 mM methylamine-0.5 M ammonium bicarbonate. Avoid heating this buffer during preparation. Heat increases the formation of cyanate in the urea that can lead to the carbamylation of the e-amino group of lysine residues. Meth-ylamine helps eliminate cyanate in urea. 

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 mechanism resembles that proposed for a phosphotriesterase (Fig. 12-24). The triesterase catalyzes detoxification of organophosphorus toxins such as parathion (Box 12-E) and seems to have evolved rapidly from a homologous protein of unknown function.721 The phosphotriesterase contains two Zn2+ ions in a dimetal center. An unusual structural feature is a carbamate group, formed from Lys 169 and C02, which provides a bridging ligand for the metal pair.721-725 A carbamylated lysine also functions in ribulose bisphos-phate carboxylase (Fig. 13-11). 

Figure 12-24 Hypothetical event in the action of a phosphotriesterase. A carbamylated lysine (lower center), as well as a water molecule, bridge the two Zn2+ ions, which are held by imidazole and aspartate carboxylate groups. The bound H20 can be deprotonated to give the HO complex shown. The substrate may displace the HO - ion from the right-hand zinc and thereby move close to the bound HO which attacks as indicated. Based on Cd2+-containing structure and discussion by Benning et al.722...

Figure 13-11 (A) Overview of the active site of spinach rubisco showing bound 2-carboxy-D-arabinitol 1,5-bisphosphate and Mg2+ and residues within hydrogen-bonding distance of these ligands. The hydroxyl groups at C2 and C3 of the inhibitor are in cis conformation. 269 Courtesy of Inger Andersson. (B) Structure of the inhibitor 2-carboxy-D-arabinitol 1,5-bisphosphate. A part of the carbamylated lysine 201 and the essential metal ion are also shown.

Jack bean urease is a trimer or hexamer of identical 91-kDa subunits while that of the bacterium Klebsiella has an (a(32y2)2 stoichiometry. Nevertheless, the enzymes are homologous and both contain the same binickel catalytic center (Fig. 16-25).435-4373 The three-dimensional structure of the Klebsiella enzyme revealed that the two nickel ions are bridged by a carbamyl group of a carbamylated lysine. Like ribulose bisphos-phate carboxylase (Fig. 13-10), urease also requires C02 for formation of the active enzyme.438 Formation of the metallocenter also requires four additional proteins, including a chaperonin and a nickel-binding protein.438 439... 

Figure 16-25 The active site of urease showing the two Ni+ ions held by histidine side chains and bridged by a carbamylated lysine (K217 ). Abound urea molecule is shown in green. It has been placed in an open coordination position on one nickel and is shown being attacked for hydrolytic cleavage by a hydroxyl group bound to the other nickel. Based on a structure by Jabri et al.i36 and drawing by Lippard.437...

A clearer picture emerged when the crystal structure of K. pneumoniae urease was determined [27], The nickel atoms in the center, Ni-1 and Ni-2, are 3.5 A apart. They are bridged by a carbamyl group, formed from C02 and a lysine residue, explaining the requirement for hydrogen carbonate in reconstitution. The other ligands are two histidines for Ni-1 and an aspartate and two histidines for Ni-2. 

Stadtman has demonstrated (42) the conversion of carbamyl phosphate and acetate to acetyl phosphate, catalyzed either by a crude lysine system or by a purified acetate kinase from C. sticklandii. ADP and... 

Homocitrulline elutes from most analyzer columns just prior to valine. Alternatively, the unreacted lysines can be converted under denaturing conditions to other acid-stable derivatives (homoarginine, methyl-lysines, carboxymethyllysines) which can be quantitated after acid hydrolysis (above and 2.12). The homocitrulline content can then be assumed to be the difference between the total lysine content and the content of the acid-stable derivative. Alkaline hydrolysis of carbamylated proteins gives quantitative conversion of homocitrulline to lysine (Stark and Smyth 1963), and this can be used to check the homocitrulline content of the protein in which the other lysines have been converted to another derivative that does not give lysine by alkaline hydrolysis (see also 3.I.2.I.). 

The extent of modification can be assessed either by using "relabeled cyanate, or for lysine by determining the homocitrulline content of the protein after acid hydrolysis. Since homocitrulline is slowly hydrolyzed to lysine, the carbamylated protein should be hydrolyzed for 24 and 48 hr, and the values for homocitrulline extrapolated with first order kinetics to zero time (Plapp et al. 1971 2.7.4). 

Figure 47-SO The major metabolic pathways for the use of ammonia by the hepatocyte. Solid bars indicate the sites of primary enzyme defects in various metabolic disorders associated with hyperammonemia /) carbamyl phosphate synthetase I, (2) ornithine transcarbamylase, (3) argininosuccinate synthetase, (4) argininosuccinate lyase, (5) arginase, (6) mitochondrial ornithine transport, (7) propionyi CoA carboxylase, (fi) methylmalonyl CoA mutase, (9) L-lysine dehydrogenase, and (10) N-acetyl glutamine synthetase. Dotted lines indicate the site of pathway activation (+) or inhibition ( ). (From Flannery OB, Hsia YE, Wolf 6. Current status of /lyperommofiemjo syndromes. Hepatology 1982 2 495-506,)...

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