Serine activating enzyme

Fluorophosphates are also highly toxic and relatively volatile. Sarin and soman are chemical warfare agents. Diisopropyl fluorophosphate (DFP) is often used by biochemists to study serine-active enzymes. Mipafox and DFP cause OPIDN in humans and experimental animals. 

A completely distinct enzyme has been found in a number of organisms, which carry out the metabolism of amino acids. In this group, a pyruvoyl group is covalently bound to the active enzyme that is produced from a proenzyme in a self-maturation process (Toms et al. 2004). The proenzyme contains a serine residue that undergoes rearrangement to an ester followed by conversion into the (3-chain of the enzyme and a dehydroalanine residne that forms the A-terminal pyruvoyl group of the a-chain. This type of enzyme has been fonnd for a number of important decarboxylations ... 

The power of the pooled GST fusion protein approach will increase as new biochemical reagents and assays become available. The development of chemical probes for biological processes, termed chemical biology, is a rapidly advancing field. For example, the chemical synthesis of an active site directed probe for identification of members of the serine hydrolase enzyme family has recently been described (Liu et al., 1999). The activity of the probe is based on the potent and irreversible inhibition of serine hydrolases by fluorophosphate (FP) derivatives such as diisopropyl fluorophosphate. The probe consists of a biotinylated long-chain fluorophosphonate, called FP-biotin (Liu et al., 1999). The FP-biotin was tested on crude tissue extracts from various organs of the rat. These experiments showed that the reagent can react with numerous serine hydrolases in crude extracts and can detect enzymes at subnanomolar... 

On intuition, a minute amount of water was added to the solvent (ethyl acetate) in the first crystallization experiment containing a molar excess of imidazole corresponding to 1, Regularly shaped crystals were formed within one hour. Such a crystal, subjected to X-ray analysis, has the structure as shown in Fig. 41 U1). Apart from the formation of the expected salt-type associate (carboxylate-imidazolium ion pair, cf. Sect. 4.2.2), two water molecules are present in the asymmetric unit of the crystal structure. This fact called our attention again to the family of serine protease enzymes, where water molecules are reported as being located in the close vicinity of the active sites 115-120),... 

The protein-based clotting process is a classic example of an enzyme cascade (see Figure 5.23). The clotting factors (which are designated with a Roman numeral, I to XIII) are synthesized in the liver and circulate in the blood as inactive precursors, strictiy, proenzymes. Most of the clotting factors are serine protease enzymes, that is they are enzymes which cleave other proteins (substrates) by a mechanism which involves a serine residue at the active site. 

The cholinesterases, acetylcholinesterase and butyrylcholinesterase, are serine hydrolase enzymes. The biological role of acetylcholinesterase (AChE, EC 3.1.1.7) is to hydrolyze the neurotransmitter acetylcholine (ACh) to acetate and choline (Scheme 6.1). This plays a role in impulse termination of transmissions at cholinergic synapses within the nervous system (Fig. 6.7) [12,13]. Butyrylcholinesterase (BChE, EC 3.1.1.8), on the other hand, has yet not been ascribed a function. It tolerates a large variety of esters and is more active with butyryl and propio-nyl choline than with acetyl choline [14]. Structure-activity relationship studies have shown that different steric restrictions in the acyl pockets of AChE and BChE cause the difference in their specificity with respect to the acyl moiety of the substrate [15]. AChE hydrolyzes ACh at a very high rate. The maximal rate for hydrolysis of ACh and its thio analog acetyl-thiocholine are around 10 M s , approaching the diffusion-controlled limit [16]. 

FIGURE 6.9 The classical pathway of complement activation is initiated by binding of Clq to antibody on a surface such as a bacterial surface. Multiple molecules of IgG bound on the surface of a pathogen allow the binding of a single molecule of Clq to two or more Fc pieces. The binding of Clq activates the associated Clr, which becomes an active enzyme that cleaves the proenzyme Cls, generating a serine protease that initiates the classical complement cascade. 

When 14C-labeled serine was fed to organisms producing histidine decarboxylase, 14C was incorporated into the bound pyruvoyl group (Fig. 14-11). Thus, serine is a precursor of the bound pyruvate. The enzyme is manufactured in the cell as a longer 307-residue proenzyme which associates as hexamers (designated n6). The active enzyme was found to be formed by cleavage of the n chains between Ser 81 and Ser 82 to form 226-residue a chains and 81-residue (3 chains which associate as (aP)6.270/271 The a chains... 

Both enzymes are inhibited by sodium borohydride and also by nitromethane. After reduction with NaB3H4 and hydrolysis, 3H-containing alanine was isolated. This suggested that they contain dehydroalanine, which could arise by dehydration of a specific serine residue.286,287 For phenylalanine ammonia-lyase from Pseudomonas putida this active site residue has been identified as S143. Replacement by cysteine in the S143C mutant also gave active enzyme while S143A... 

Vithayathil et al. (34%). In 0.5 M HC1 at 30° RNase-A undergoes structural alterations which can be detected chromatographically at neutral pH. However, all the products are equally active enzymically, and no reaction would have been detected by assay. At pH 11.0 even more involved structural changes take place quite rapidly. Irreversible alkaline denaturation takes place at higher pH and is very rapid at 13. Here the activity loss is accompanied by marked spectral changes indicating reactions such as / elimination at cystine or serine residues (343). A temperature-induced isomerization at neutral pH has been reported by French and Hammes. This is discussed in a later section on nucleotide binding. 

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