Inverse substrates

Table E.5.2. Inverse substrate concentration and inverse enzymatic rate calculation with and...

K, Tanizawa, Y Kasaba, and Y. Kanaoka- Inverse substrates" for trypsin. Efficient hydrolysis of certain esters with a cationic center in the leaving group J. Am. Chem. 

Design of Trypsin Substrates of a New Type — Inverse Substrates.98... 

Inverse Substrates for Trypsin-Like Enzymes — Medicinal Applicabilities 101... 

This present article surveys the recent development of biospecific compounds which interact with active sites of enzymes. These compounds are classified according to their mode of interaction. The characteristic features of interaction are discussed and the molecular basis for the design of the specific compounds of each type is considered. Significance of the enzyme-specific compounds in basic research and in the application of chemotherapeutics is exemplified. The development of inverse substrates , specific compounds for trypsin and trypsin-like enzymes of a new type, is also described. The basic idea for the design of inverse substrates and their applicabilities are discussed. 

The reaction process of trypsin-catalyzed hydrolysis of the inverse substrates is illustrated in Fig. 4. Here the process is compared to that of normal-type substrates. After specific binding and efficient acylation, the site-specific amidinophenyl moiety is cleaved (leaving group) to give the acyl enzyme in a very specific manner. As a result, inverse substrates are expected to be applicable as a general method for specific introduction of any acyl group of non-specific structure into the trypsin active site. 

For the first time, inverse substrates provide a general method for the specific introduction of an acyl group into the trypsin active site without recourse to cation-containing acyl compounds. The preparation of various new acyl enzymes is expected to lead to the discovery of novel features of the enzymatic reaction mechanism. In addition, any desired reporter groups might be specifically introduced into the trypsin... 

Fig. 4. Reaction sequences of trypsin with normal type and inverse substrates. The hydroxyl function and negative charge represent the catalytic residue (Ser-195) and the binding residue (Asp-189) at the active site, respectively. The acyl trypsin-ligand complex (low right) formed in the presence of a cationic compound...

It is of special value to extend the inverse concept further to trypsin-like enzymes. Inverse substrates of these biologically important enzymes could be candidates for clinically useful substances. In the following sections, various aspects of the applicabilities are briefly described. 

An additional characteristic of inverse substrates is also shown in Fig. 4. The acyl enzyme formed from the inverse substrate lacks a site-specific cationic residue with... 

Table 4. Kinetic parameters for the trypsin-catalyzed hydrolysis of inverse substrates at pH 8.0,25 °C...

It is well known that the specificity of an enzyme such as thrombin and plasmin is very close to that of trypsin. In this respect, inverse substrates for trypsin also are expected to be susceptible to the catalysis by these enzymes. In the kinetic analysis of trypsin-like enzymes toward p-amidinophenyl esters, it was found that the inverse concept is also applicable to thrombin, plasmin, urokinase, kallikrein, and trypsins from various origins 74 - 75). These enzymes are not distinctively different from bovine-... 

The active site structure of trypsin-like enzymes is considered to be very similar to that of bovine trypsin, yet little is known about them. Refinement of these structures is important also for the purpose of designing physiologically active substances. With a view to comparing the spatial requirements of active sites of these enzymes, dissociation constants of the acyl enzyme-ligand complex, K-, which were defined before, were successfully analyzed By taking advantage of inverse substrates which have an unlimited choice of the acyl component, development of stable acyl enzymes could be possible. These transient inhibitors for trypsin-like enzymes could be candidates for drugs. In this respect, the determination of the deacylation rate constants for the plasmin- and thrombin-catalyzed hydrolyses of various esters were undertaken 77). 

A new approach to thrombosis therapy using acyl plasmins has been reported by Smith et al.78). Acyl plasmin is catalytically inert and unable to react with plasma inhibitors but still can bind to a fibrin clot. Thus, after the administration, acyl plasmin can circulate without being trapped by the inhibitors and can come into contact with fibrin. Deacylation may then occur to give a fibrin-plasmin complex and this active enzyme is expected to lead to fibrinolysis. The preparation of acyl plasmin of appropriate stability was realized by using the general procedure for the specific synthesis of an acyl enzyme — the inverse substrate method. 

Among a number of experimental results in which the kinetic behavior of protelytic enzymes toward a variety of synthetic substrates and inhibitors have been tested, some seemingly irrational enzymatic responses were observed. Example of these responses will be discussed from the viewpoint of the imperfectness of the enzymatic recognition. The existence of inverse substrates might be due to such an imperfectness or allowance in the recognition rigidity of the enzyme. 

Further search for inverse substrates other than /7-amidinophenyl esters has been carried out and it has been found that esters derived from p-amin omcthylphcnol and /7-guanidinophenol were also eligible as a substrate of trypsin and trypsin-like enzymes 75 86). We have also found that trimethylaminobutanoic acid p-nitrophenyl ester is an inverse substrate for butyrylcholinesterase 87-88(. Application of the inverse concept to thiol enzymes was also successful p-amidinophenyl esters were found to be substrates for clostripain 74), a thiol enzyme with trypsin-like specificity. Although the design of inverse-type substrates seems not always possible for a variety of hydrolytic enzymes, this new concept could provide potential means for certain enzymes to both fundamental study and application. 

CSVT allows short experiments and easy determination of the growth rate. In this technique, a source and a substrate are placed at very short distance ( 1mm). The experiments were achieved under a hydrogen flow of 1 l/min at two source temperatures, 550 and 600°C. In Fig. 1 is pictured the instantaneous growth rate as a function of the inverse substrate temperature for these two source temperatures. The solid and dashed lines correspond to a fit using a theoretical model. ... 

Figure 5.28 ATjnv (0 Szo) as a function of inverse substrate separation l/sjo in the limit of vanisliing fluid density [see Eq. (5.219)]. According to mean-field theory, data points should fall on a straight line through the origin (see text). The straight solid line is a fit to the data points using only entries for 1/Szo = 0,0.01.

Tam JP, Lu Y, Liu CF et al. (1995) Peptide synthesis using unprotected peptides through orthogonal coupling methods. Proc Natl Acad Sci USA 92(26) 12485-12489 Thormann M, Thurst S, Hofmann H et al. (1999) Protease-catalyzed hydrolysis of substrate mimet-ics (inverse substrates) a new approach reveals a new mechanism. Biochemistry 38(19) 6056-6062... 

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