Synthetic substrate

The pH dependence of HIV-1 protease has been assessed by measuring the apparent inhibition constant for a synthetic substrate analog (b). The data are consistent with the catalytic involvement of ionizable groups with pK values of 3.3 and 5.3. Maximal enzymatic activity occurs in the pH range between these two values. On the basis of the accumulated kinetic and structural data on HIV-1 protease, these pK values have been ascribed to the... 

In vitro and ex vivo studies have shown that FATPs transport LCFAs and very long-chain fatty acids (VLCFAs) but no medium-chain fatty acids, fatty acid esters, or lipid-soluble vitamins [4]. LCFA transport is inhibited by prior protease treatment. Synthetic substrates for FATPs include 14C-labeled fatty acids and the fluorescently labeled fatty acid analogue C1 -BODEP Y-Cl 2. Using the latter substrate, differences in fatty acid uptake kinetics between FATP expressing 3T3 LI adipocytes and 3T3 LI fibroblasts, which are devoid of FATPs, can be readily appreciated (Fig. 2). 

Silverberg M, Dunn J, Garen L, Kaplan A Autoactivation of human Hageman factor. Demonstration utilizing a synthetic substrate. J Biol Chem 1980 255 7281-7286. 

Recent evidence from our laboratory shows that the supernatants of HHMC activated by anti-IgE can convert a synthetic substrate of big endothelin to endothelin 1. The latter observation is particularly important because endothelin 1 is a potent bronchoconstrictor in allergic subjects [30]. 

Overview of Calcium Carbonate Crystallization by Synthetic Substrates... 

Thus, if we can apply the type of asymmetric decarboxylation reactions mentioned above to synthetic substrates, unique asymmetric reactions and C—C bond-forming reactions will be realized which are otherwise difficult to be realized. 

This enzyme was shown to be specific for xylan oligomers and small acetylated synthetic substrates. Many characteristics have been published recently about this type of enzyme, purified from Trichoderma reesei, and A. oryzae [6], and a different A. n/ger preparation[7]... 

Another interesting target for this type of inhibitors is the dipeptidyl peptidase IV (DPP IV). This exodipeptidase, which can cleave peptides behind a proline residue is important in type 2 diabetes as it truncates the glucagon-like peptide 1. Taking into account the P2-Pi( Pro)-P,1 cleavage and the requirement for a free terminal amine, the synthesis of a suicide inhibitor was planned. It looked as if the the e-amino group of a P2 lysine residue could be cyclized because of the relative little importance of the nature of the P2 residue on the rate of enzymatic hydrolysis of known synthetic substrates. Therefore, anew series of cyclopeptides 11 was synthesized (Fig. 11.8). 

Lindley, H. (1956) A new synthetic substrate for trypsin and its application to the determination of the amino acid sequence of proteins. Nature (London) 178, 647. 

Affinity capture-release electrospray ionization mass spectrometry (ACESIMS) is another recently introduced technique for quantification of proteins, and to date has most often been applied to clinical enzymology.60 The product conjugates of the enzymatic reaction between the synthetic substrate and targeted enzyme are captured by immobilized affinity reagents, purified, released into solution, and analyzed by ESI-MS. 

Affinity complexation — Many proteins have affinities for other molecules that can be exploited to alter their retention characteristics in IEC. For example, some enzymes may be combined with synthetic substrates, cofactors, or products.1315 The same principle can be applied to other protein/receptor systems. One well-characterized example is the change in chromatographic behavior of fructose 1,6-diphosphatase in the presence of its negatively charged substrate... 

Serine hydroxymethyl transferase catalyzes the decarboxylation reaction of a-amino-a-methylmalonic acid to give (J )-a-aminopropionic acid with retention of configuration [1]. The reaction of methylmalonyl-CoA catalyzed by malonyl-coenzyme A decarboxylase also proceeds with perfect retention of configuration, but the notation of the absolute configuration is reversed in accordance with the CIP-priority rule [2]. Of course, water is a good proton source and, if it comes in contact with these reactants, the product of decarboxylation should be a one-to-one mixture of the two enantiomers. Thus, the stereoselectivity of the reaction indicates that the reaction environment is highly hydro-phobic, so that no free water molecule attacks the intermediate. Even if some water molecules are present in the active site of the enzyme, they are entirely under the control of the enzyme. If this type of reaction can be realized using synthetic substrates, a new method will be developed for the preparation of optically active carboxylic acids that have a chiral center at the a-position. 

The purified enzyme was most active at pH 6.0 and stable at 40°C for 30 min in the pH range of 6.5 to 8.0. The enzymatic activity was strongly inhibited by the addition of metal ions and some chemicals but not by D-mannose as shown in Table II and III. Among of the synthetic substrates so far tested, the enzyme did not hydrolyze any of... 

Figure 3. pH profiles of wild type and two variant subtilisins. Activity was assayed with synthetic substrates as described (2) at the indicated pH. 

These studies with acetamidoglycosides provide support for the plausibility of mechanisms for lysozyme in which nucleophilic attack by the neighbouring group takes place. The synthetic substrates developed for lysozyme which lack this group (Raftery and Rand-Meir, 1968) render unnecessary any postulate of involvement of the 2-acetamido-group, but, of course, do not rule out the possibility. 

Riester, D., Wegener, D., Hildmann, C. and Schwienhorst, A. (2004) Members of the histone deacetylase superfamily differ in substrate specificity towards small synthetic substrates. Biochemical and Biophysical Research Communications, 324, 1116-1123. 

Five- to six-month-old tobacco plants (Nicotiana tabacum var. Samsun) grown in a glasshouse at 20°C were used for this study. Commercial synthetic substrates employed both for histochemical and biochemical assays were guaiacol, p-phenylenediamine-pyrocatechol (PPD-PC), 3-3 di-aminobenzidine (DAB), tetramethylbenzidine (TMB) and syringaldazine. Isopropylamine and monosodium salts of ferulic acid were also used as substrates as well as their / -fluorinated analogues substituted with a fluorine atom on the / -carbon (Fig. 1). Histochemical observations were done on hand-made transverse sections of fresh tobacco stems. Biochemical assays were performed separately on bark (inner cortical parenchyma, phloem and fibres) and xylem fractions. Technical data of incubation, enzyme extraction, spectrophotometric and electrophoretic assays were given elsewhere (5-7). Synthesis of fluorinated compounds was performed as previously described (4). 

Thus ferulic acid, which is not in vivo a natural substrate for peroxidases involved in lignification processes, can be oxidized not only in vitro but also in situ, i.e., in the normal, biological environment of the enzyme. Furthermore, the oxidation seems to be limited to the walls of lignifying cells. This restricted localization has been described only in the case of syringaldazine, a synthetic substrate closely related to cinnamic compounds... 

Inhibition of Commercial Synthetic Substrates with Salts from Ferulic and j3-Fluoroferulic Acids. Table I summarizes the results obtained on stem sections incubated in a medium containing one of the usual commercial substrates and a salt of ferulic or / -fluoroferulic acid. Three types of interactions could be observed ... 

Similarly, we could create a mutant in part of the polyfunctional protein and allow the synthetic substrate to be accepted by the functional parts and carried through to produce a completely different compound. The benefit of this type of manipulation is that synthetic procedures that are very difficult for chemists to do... 

In preparing these various libraries, extensive use is made of solid phase synthetic methods. These methods are all derived from the solid phase peptide synthesis (SPPS) method developed by Merrifield in 1963. When performing a large number of syntheses, it is preferable to perform the synthetic steps on a solid bead rather than completing the entire synthesis in the solution phase. The solid-phase technique makes byproduct removal and final compound purification easier. The organic chemistry literature contains a wealth of different types of solid-phase supports and novel linkers for attaching the synthetic substrate to the bead. 

The pharmaceutically very interesting dibenzocyclooctadiene lignans, such as steganacin, steganone and schizandrin8-10, constitute ideal synthetic substrates for this method since they have the required oxygenation pattern. 

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