Pseudo substrate

The role of the serine residue in hydrolysis was further examined using pseudo-substrates, e.g. p-nitrophenylacetate—substrates which were only very slowly utilized by the enzyme. The p-nitrophenyl group was slowly released and the acyl group became attached to the same serine in hydrolases which had been detected by DIPF (Kilby and Youatt, 1954). Mechanisms for peptide and ester hydrolysis were therefore proposed in which the acyl group became transiently and covalently bound to serines in catalytic sites (see Hartley et al. 1969). 

The structural analysis of the trypsin inhibitor from bovine pancreas (BPTI) in complex with trypsin shows that the inhibitor occupies and blocks the substrate binding pocket in a highly complementary maimer (fig. 2.9). In the trypsin-BPTI complex, the catalytically essential Ser-OH of trypsin contacts a CO group of the inhibitor in a manner very similar to the tetrahedral transition state of amide or ester bond hydrolysis (see fig. 2.9b). The inhibitor can be likened to a pseudo-substrate and, as such, is bound with high affinity. The cleavage of the peptide bond is, however, not possible due to other circumstances, such as the fact that water is prevented from reaching the active site with the inhibitor boimd. 

In a subsequent report, in 2005 [55], the same group described the preparation of imprinted polymer capable of oxidising alcohols and alkanes with 2,6-dichlor-opyridine /V-oxide (86) without mineral acid activation. The polymer was imprinted with a ruthenium porphyrin complex (87) using the diphenylmethana-mine (88) as pseudo-substrate template in order to achieve a shape of the cavity complementary to the substrates, diphenylmethane (89) and diphenylmethanol (84). The reaction, carried out with the imprinted polymer on the diphenylmethanol as substrate, showed a rate enhancement 2.5 higher than with the non-imprinted polymer. In the same conditions, but with diphenylmethane and... 

There are just a few studies of the use of caspase inhibitors to prevent apoptosis. Most studies concentrate on the expression of proteins of the IAP family (XIAP being the most noticeable) and the viral components p35 and CrmA (Vives et al., 2003a). CrmA, encoded by the smallpox virus, is a pseudo-substrate for serine and cysteine proteases. It inhibits caspases 1, 8, and 10 in several cell types (Sauerwald et al., 2003). p35 is a wide-spectrum caspase inhibitor encoded by baculoviruses, and it also behaves as a pseudo-substrate, inhibiting caspases 1, 3, 6, 7, 8, and 10 (Zhou et al., 1998). XIAP is the most potent member of the IAP family. It is found in the mammalian genome and is responsible for the inhibition of caspases 3, 7, and 9 (Sauerwald et al., 2002). An increased protective effect is found in CHO and HEK-293 cells expressing a XIAP mutant resistant to degradation (XIAP-BIR123) when compared with the wild-type protein (Sauerwald et al., 2002). 

Once a protein structure has been solved, the study of the association of small molecules with the protein may be accomplished relatively easily by means of difference Fourier syntheses. The method has been widely applied in the study of binding of inhibitors and pseudo-substrates to a large number of proteins and has provided the means by which active and allosteric sites may be located. It is assumed that the small ligand does not change the unit cell or perturb the protein substantially, and that the protein phases are approximately equal to those for the protein and ligand. Small changes in conformation can be distinguished as in conventional difference syntheses. The coefficients used are... 

In many protein kinases, particularly those which are under the control of second messengers, the regulatory domain contains a pseudo-substrate sequence which binds to the catalytic site and thus prevents access of external substrates to the catalytic site. Binding of the second messengers to the regulatory domain relieves this... 

The first strategy consists in attaching a non-polymerizable imprinting ligand (which acts as a pseudo-substrate) to the metal center as a template. The metal is then incorporated into the cross-linked backbone by one or more polymerizable ligands (most frequently containing a styrene side chain to allow its incorporation into organic... 

EXAMPLE 5.15 X-ray analysis of crystals of the pancreatic exopeptidase carboxypeptidase A, with a bound pseudo substrate (z. false substrate that is not degraded by the enzyme, i.e., an inhibitor) indicates that the susceptible peptide bond is twisted out of the normal planar configuration that is usually seen with peptide bonds (Chap. 4). This distortion leads to a loss of resonance energy in the bond, and enhances its susceptibility to hydrolytic attack. 

Figure 11 Generation of immobilized metal catalysts with a substrate pocket by imprinting with a pseudo-substrate (a) attachment of the pseudo-substrate (b) polymerization (c) selective cleavage of the pseudo-substrate (d) removal of the pseudo-substrate and the metal ion (e) addition of the catalytically active metal ion.

In extension of this work, a ehiral rhodium(III) eomplex having a methyl-phenyl-phosphinato ligand was synthesized [24]. This pseudo-substrate mimies the... 

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