Active serine residue

The first step, which is called the acylation reaction, involves a formation of an acyl-enzyme where the RC(0 )X group is covalently bound to the specially active serine residue and the XH group is expelled from the active site. The second step, which is called the deacylation step, involves an attack of an HY group on the acyl-enzyme. Here we concentrate on the acylation step which is the reverse of the second step when X and Y are identical. 

Figure 51-3. Diagrammatic representation (notto scale) of prothrombin. The amino terminal is to the left region 1 contains all ten Gla residues. The sites of cleavage by factor Xa are shown and the products named. The site of the catalytically active serine residue is indicated by the solid triangle. The A and B chains of active thrombin (shaded) are held together by the disulfide bridge.

Serine proteases (SP) are a family of enzymes that use a uniquely activated serine residue in the substrate-binding pocket to catalytically hydrolyze peptide bonds [66], SP carry out a diverse array of physiological functions, of which the best known are digestion, blood clotting, fibrinolysis, fertilization, and complement activation during immune responses [67], They have also been shown to be abnormally expressed in many diseases including cancer, arthritis, and emphysema [42, 43, 67-70],... 

Serine proteases can be classified according to their homologies in amino acid sequence, which provide a measure of evolutionary relationships, or according to their specificity. The most readily determined significant feature of the sequence is the sequence around the active serine residue. This residue is easy to identify, for example, by treating the enzyme with P-labelled DFP. Table I summarizes the present data at the tripeptide level. 

Table I. Sequences at the Active Serine Residue of Serine Proteases...

To foUow this chemoenzymatic approach, the synthetic substrates must be transferred onto the catalyticaUy active serine residue of the TE domain. This transfer can either be done directly or with the help of a PCP domain. In the natural system, translocation is realized by the interaction between the PCP and the TE domain. The substrate, which is bound to the 4 Ppan cofactor of the PCP domain as a thioester, acylates the hydroxyl group of the serine. Chemically speaking, the acylation of the TE is a result of a iran -esterification. When using TE domains, the substrate provided in trans must also have an appropriate acylation potential. Several key techniques have been developed to covalently attach synthetic substrates to PCP and TE domains. In the first method, the relaxed substrate specificity of the 4 Ppan transferase Sfp is used to load acyl moieties... 

Not all proteases utilize strategies based on activated serine residues. 

In a recent study, a serine-phospholipid-selective phospholipase A has been purified and cloned its cDNA from rat platelets, which secrete two types of phospholipases upon stimulation (71). The purified enzyme from extracellular medium of activated rat platelets, yielded a 55-kDa protein band on SDS-polyacrylamide gel electrophoresis. The presence of active serine residues was confirmed by labeling the 55-kDa protein with pHJDiisopropyl fluorophosphate, an inhibitor of the enzyme. Based on cDNA for the enzyme cloned from a rat megakaryocyte cDNA library, the 456-amino acid... 

Diisopropylfluorophosphate, which irreversibly binds to active serine residues on some hydrolytic enzymes, is an example of this type of inhibitor, decreases because some enzyme is completely removed from the system. (Remember, = j>rE]i.) An irreversible inhibitor can be dis-... 

The DFP reacts with serine residues at the active site. The minimum molecular weight of the enzyme is that which contains one mole of active serine residue. The number of moles of P bound to the enzyme equals the number of moles of active serine (assuming the reaction went to completion). 

The enzyme has an active serine residue and is specific for long-chain acyl derivatives. 

YNot all proteases utilize strategies based on activated serine residues, Classes of proteins have been discovered that employ three alternative approaches to peptide-bond hydrolysis (Figure 9.17). These classes are... 

Boronic acid derivatives have been shown to inhibit -lactamases [168]. The boronic acid function, which is isoelectronic with a protonated carboxyl group, is presumed to block enzymatic activity by forming a stable tetrahedral borate complex with the active serine residue. Boronic acids 319, structurally related to penicillin G and methicillin, were synthesised in three steps from dibutyl iodomethaneboronate [169] (Scheme 97). They are inhibitors of P-lactamase I from B. cereus. There are presently no reports of boronic acid derivatives acting as inhibitors of bacterial D,D-peptidases. 

Partial reactions for the yeast Type I synthetase were studied in a classic series of experiments by Lynen (1967). Peptides capable of partial reactions have been isolated from the yeast and chicken liver enzymes (cf. Wakil et ai, 1983). Particular interest has been focused on the thioesterase. This enzyme, more easily isolated by partial proteolysis than those for most of the other partial reactions, is important in at least partly regulating chain length termination. Thioesterases isolated from several animal fatty acid synthetases have very similar amino acid sequences around the active serine residue (Poulose et al, 1981). The medium-chain fatty acids produced by synthetases from some mammary glands appear to be due to thioesterase II (a second thioesterase) (Libertini and Smith, 1978). 

The boundaries of the model represent the space available for the accommodation of the substrate. The important binding regions which determine the selectivity of the reaction are two hydrophobic pockets (Hl and Hs, with L = large and S = small) and two pockets of more polar character (Pp and Pb, with F = front and B = back). The best fit of a substrate is determined by positioning the ester group to be hydrolyzed close to the hydrolytically active serine residue and then arranging the remaining moieties in the H and P pockets. 

Partial sequences of homologous proteolytic (Al and esterolytic (B) enzymes from the region about the active serine residue ... 

Serine hydrolases hydrolases which have a cata-lytically active serine residue in their active center, e. g. trypsin, chymotrypsin A, B and C, thrombin and B-type carboxylic acid esterases. See Serine proteases. 

An exceptionally reactive serine residue has been identified in a great number of hydrolase enzymes, e. g., trypsin, subtilisin, elastase, acetylcholine esterase and some lipases. These enzymes appear to hydrolyze their substrates by a mechanism analogous to that of chymotrypsin. Hydrolases such as papain, ficin and bromelain, which are distributed in plants, have a cysteine residue instead of an active serine residue in their active sites. Thus, the transient intermediates are thioesters. 

In particular, thiolactonization is much lower in rate. The orientation is thus less favorable and this is expected because of the distortion in the overall shape of electron orbitals. Similarly, if the hydroxyl group of the active serine residue of the enzyme subtilisin is replaced by a —SH function, the activity of the protease is drastically reduced. This substitution (O -> S) has a considerable effect (lowering) on the acyl-enzyme deacylation rate constant (69). Koshland argues that such inhibition would be expected if orbital orientation were of great importance. 

Diisopropylphosphorofluoridate (DFP, Section 4.4) is also an active-site irreversible inhibitor that blocks the active serine residue of serine proteases. It is easy to show that the inhibition is irreversible since exhautive dialysis still produces an inactive enzyme. 

The active site of chymotrypsin strongly resembles that of trypsin Again there is a catalytically active serine residue which reacts with the inhibitor diisopropylfluorophosphate and whose immediate vicinity exhibits the same amino acid... 

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