Carbamylated enzyme

In the interaction of isoflurophate with AChE, a phosphorylated intermediate is formed and fluoride is released. An important characteristic of the organophos-phate-induced inhibition is that the bond between the phosphate and the enzyme is very stable. While the regeneration of most carbamylated enzymes occurs with a half-life of minutes or hours, the recovery of a phosphorylated enzyme is generally measured in days. These agents are referred to, therefore, as irreversible inhibitors. 

Carbamates effect the reversible carbamylation of acetylcholinesterase, permitting accumulation of acetylcholine at cholinergic neuroeffector junctions (muscarinic effects), at the myoneural junctions of skeletal muscle, and in the autonomic ganglia (nicotinic effects). CNS function is also impaired. However the relatively large dissociation constant of the carbamyl-enzyme complex indicates that it dissociates more readily than does the organophosphate-enzyme complex, mitigating the toxicity of the carbamate pesticides. The reversibility of the carbamyl-enzyme complex affects (limits) the utility of blood enzyme measurements as a diagnostic tool. 

Carbamylated enzymes that carry a much longer (C=0)-N-substituent, have a considerably longer off-rate. The N-heptyl analogue of physostigmine (see below) has a tVi of 11 days, but this was attributed to a hydrophobic interaction between the chain and the enzyme. 

Irreversible inhibitors are effectively esteratic site inhibitors which, like true substrates, react with the hydroxyl group of serine at the catalytic active site. Such inhibitors, sometimes referred to as acid-transferring inhibitors, include the organophosphates, the organo-sulfonates, and the carbamates. All form acyl-enzyme complexes which, unlike substrate-enzyme intermediates, are relatively stable to hydrolysis. Indeed, the phosphorylated enzyme intermediates have half-lives from a few hours to several days (A12), whereas the sulfonated or carbamylated enzyme complexes have much shorter half-lives—several minutes to a few hours. Several strong lines of direct evidence point to the formation of an acyl complex—the isolation of phosphorylated serine from hydrolysates of horse cholinesterase (J2), complex formation and carbamylation (02), and the sulfonation of butyrylcholinesterase by methanesulfonyl fluoride in the presence of tubocurarine and eserine (P6). 

Derivatives of aminoformic acid react with ChE similarly to OPC, i.e. in two stages with establishing a covalent bond. A degree of the anti-cholinesterase action of carbamates depends upon strength of the complexes formed. Evidently, the phosphorylated ChE becomes disabled for a more prolonged period, as compared with the carbamylated ChE. Rapidity of the initial activity restoration of the enzyme inhibited by carbamates is determined, respectively, by the carbamylated enzyme hydrolysis speed, which depends upon the inhibitor structure. Carbamy-... 

The half-lives and the corresponding k+3 values are dependent on the structure of, and therefore the source of, the enzyme and the structure of the group attached to the serine residue of the enzyme. Note the tremendous difference between the acetylated enzyme and the enzyme s phosphate and carbamate adducts. The half-lives are generally lower for the carbamylated enzymes but long enough to cause serious poisoning. 

The carbamate anticholinesterases such as PB bind reversibly with ChE enzyme, yet spontaneously reactivate relatively rapidly. However, nerve agents (organophosphate compounds) bind with the ChE irreversibly and form a much more stable phosphory-lated enzyme (ChE-OP) complex. PB binds to peripheral ChE at anionic and esteratic sites and thus carbamylates the enzyme. The carbamylated enzyme sites cannot bind with nerve agents. In the meantime, some of the nerve agents are hydrolyzed to inactive metabolites by nonspecific hydrolases. The decarbamylation of the ChE takes place at the alcohol moiety on the esteratic site, regenerating the ChE enzyme to sustain life. 

OPs and carbamates (CMs) react covalently as pseudosubstrates at the catalytic center of a variety of serine hydrolases. Contrary to true substrates, the rate of hydrolysis of the carbamylaled enzymes is slow (minutes to hours) and that of phosphorylated enzymes is extremely slow (hours to... 

When aryl carbamate AChEls, such as physostigmine and its analogues, bind to the catalytic site of AChE, hydrolysis of the carbamate occurs. This transesterifies the serine residue with carbamic acid, forming what is termed a carbamylated enzyme. The rate of carbamylation follows the order of carbamic acid esters > methylcarbamic acid esters > dimethylcarbamic acid esters (40). 

Regeneration of active AChE by hydrolysis of the carbamylated enzyme is much slo er than hydrolysis of fhe acefylafed enzyme. The rafe for hydrolytic regeneration of the carbamylated AChE is measured in minutes (e.g., the half-life for mefhyl carbamylafed enzyme is -15 minufes) fhe rafe of hydrolytic regeneration of acetylafed AChE is measured in milliseconds (e.g., fhe half-life for acefylafed enzyme is -0.2 milliseconds). Despite the longer time required to regenerate the carbamylated enzyme, the active form of AChE evenfually is regenerafed. Therefore, fhese inhibitors are considered to be reversible. 

Rivastigmine is a centrally selective, arylcarbamate AChEI that was approved in 2000 for oral administration in the treatment of AD. It has an elimination half-life of 1.4 to 1.7 hours but is able to inhibit AChE for up to 10 hours. Because of the slow dissociation of the carbamylated enzyme, it has been referred to as a pseudo-irreversible AChEI (47). Like donepezil, rivastigmine exhibits a low level of hepatotoxicity. It is rapidly and extensively hydrolyzed in the CNS by cholinesterase with minimal involvement of CYP450. The phenolic metabolite is excreted primarily via the kidneys. 

Partial protection by bicarbonate may indicate that the carbamylated enzyme is more resistant to photoinactivation. Although non-substrate bicarbonate does bind to the active site, and actually competitively inhibits RuBP binding (6), anions such as formate, and perhaps acetate would be expected to behave similarly. The lack of protection by these compounds (5) suggests that the mode of bicarbonate protection may be due to carbamylation. The lack of any effect by Mg+ suggests that it is the binding of CO2 (or bicarbonate) that confers resistance to photomodification, and not the formation of the active ternary complex enzyme C02 Mg+. ... 

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