Secondary binding sites

Tetracycline has a secondary binding site in the H27 switch region that may also be fimctionally significant. The dtug binds at the interface of the three domains of 16S rRNA, close to helix 44 and between helices 11 and 27. As with the primary binding site, contacts are made from the hydrophilic face of the dtug to the backbone of 16S rRNA. In this binding site, tetracycline may function to stabilize the ram state. 

Primary- and secondary binding sites with different specificity exist on the S-layer protein [122]... 

The use of rhodium catalysts for the synthesis of a-amino acids by asymmetric hydrogenation of V-acyl dehydro amino acids, frequently in combination with the use of a biocatalyst to upgrade the enantioselectivity and cleave the acyl group which acts as a secondary binding site for the catalyst, has been well-documented. While DuPhos and BPE derived catalysts are suitable for a broad array of dehydroamino acid substrates, a particular challenge posed by a hydrogenation approach to 3,3-diphenylalanine is that the olefin substrate is tetra-substituted and therefore would be expected to have a much lower activity compared to substrates which have been previously examined. 

In fact, the majority of the radiolabel was recovered in the small C-terminal fragment containing TM-6 and TM-7. It delivered biochemical evidence that the aryloxyalkyl side-chain of salmeterol was positioned in that region and represented a secondary binding site, while the pharmacophore portion of the compound bound to the expected catechol site. 

The results of deuterium labeling experiments shown in Scheme 37 clearly show the operation of a monohydride mechanism in the BINAP-RuOI) catalyzed hydrogenation of unsaturated carboxylic acids. However, with many olefinic substrates with a neutral, rather than anionic, secondary binding site, the products exhibit a similar degree of isotope incorporation at the two hydrogenated centers (Scheme 39). The out-... 

Figure 9 shows the effects of warfarin on the inhibition by phenylbutazone and Figure 10 shows the effects of phenylbutazone on the inhibition by warfarin. The presence of either at equal concentration with HSA eliminates the plateau otherwise observed in the inhibition curve of the other. These results clearly seem to indicate a common noninhibitory site for these compounds. Thus the high-affinity binding site (the noninhibitory site) and the secondary binding site (the inhibitory site) for these compounds are the same. 

Fig. 7.1 The tetramer of eco bound to a serine protease. Visualized as a cartoon of the canonical protease and eco interaction (a), and (b), as two views of the three dimensional solution of D102N trypsin in complex with eco [3]. Each eco molecule has three protein-protein interaction surfaces. The C-terminus forms an anti-parallel p ribbon to complete the ecotin dimer interface. The 80 s and 50 s loops form the primary binding site by interacting with the protease at the active site cleft in a sub-strate-like y -sheet conformation. The 60 s and lOO s loops of eco form the secondary binding site by interacting with the C-termi-nal a-helix of the protease. Note that each eco molecule contacts both of the protease molecules. Two eco molecules (black and medium grey) form a pair of interactions each with two protease molecules (light grey). The catalytic triad residues Ser-195, Asp-102 and His-57 are in black ball and stick representation. This figure was made with Molscript [37] and Raster 3D [38].

As far as the fungal proteases are concerned, studies in Frutons laboratory (75) and our own laboratory (76) have provided evidence that secondary binding sites are equally or more important for increased... 

They found evidence for a secondary binding site requirement although the catalytic efficiency of chymosin and the fungal enzymes on the same substrates was lower than that of pepsin, in analogy to our findings for penicillopepsin. 

Although this kind of comparison has obvious limitations, it does give a definite indication of a relationship among the acid proteases. The basis of this relationship may be the secondary binding site requirement. 

In an attempt to find out more about the nature of the secondary binding site in penicillopepsin. Mains et al. (76) analysed the nature of the amino acid side chains at positions removed from the sensitive peptide bond. An abbreviated summary of the results is given in Table VII. The number of hydrophobic and hydrophilic side chains respectively are listed for four amino acids on either side of every peptide bond broken in the B-chain of insulin and in glucagon. The positions are numbered Pi, P2, P/, P2 etc. as defined by Berger and Schechter (103). The choice of four positions was taken from the Frutons work (73) on the specificity of pepsin and the eflFect of chain length on catalytic efficiency. The largest eflFects were observed with substrates having three to four... 

The possibility that hydrolysis proceeds without the formation of covalent intermediates has been mentioned by Fruton (73) and must be given serious attention in future studies. Because of the very large eflFects of the secondary binding sites on the catalytic efficiency, it is essential that these studies be carried out with good substrates. Silver and Stoddard (120) suggest that hydrolysis of small substrates proceeds without an amino intermediate. However, they did not consider an acyl intermediate. 

Several protease inhibitors are competitive, and they bind in the protease active site, but also they have secondary binding sites outside the active site, which are critical to inhibition. Exosite binding provides two major benefits 1) It increases the surface area of the interaction, which leads to a greater affinity, and 2) it can provide a greatly increased amount of specificity. 

Arolas JL, et al. Secondary binding site of the potato carboxypepti-dase inhibitor. Contribution to its structure, folding, and biological properties. Biochemistry 2004 43 7973-7982. 

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