Protein-substrate binding

PBPb Bacterial periplasmic substrate-binding proteins E(M)AB 0(0) 0(0) 1GGG... 

PBPe Eukaryotic homologs of bacterial periplasmic substrate binding proteins E(MP) 0(0) 8(8) 1GR2... 

Hightower, K.E., Huang, C.C., Casey, P.J., and Fierke, C.A. (1998). H-ras peptide and protein substrates bind protein farnesyltransferase as an ionized thiolate. Biochemistry 37 15555-15562. 

Biological processes involving protein-protein recognition are of special interest since they are the basis of many relevant events in the cellular machinery. Enzyme-substrate binding, protein membrane signaling, immunologic response, and protein quaternary structural changes caused by cofactors or allosteric modulators are only a few of the examples that show the importance of protein-protein interactions at the molecular level. 

These approaches have all depended on one or more characteristics of individual transport systems that are not common to all systems the transport proteins must be removable from the membrane by osmotic shock, or be inducible, or retain binding activity upon solubilization from the membrane or removal by osmotic shock. An alternative that depends on a transport system having specificity for a particular substrate, or class of substrate, and high affinity for the substrate (s) is affinity labeling. A radioactive affinity label could, in principle, be used to specifically label the substrate binding proteins of a transport system and the label could be used as a marker to follow the purification of the protein. The experience gained could then be applied to isolation of the potentially active, unlabeled protein. 

The arrangements of the protein molecules in the crystals, i.e. the symmetries of the crystals, determine the patterns of diffraction, and the structures of the protein molecules are reflected in the intensities of all diffraction intensities. Therefore similar arrangements or structures of proteins molecules should eontribute similar diffraction patterns or intensities. In principle, from the known structures, the locations and translations of similar proteins inside different crystal cells can be deduced, and then the target proteins can be replaced by known model proteins to obtain initial models for the unknown crystal structures. This is the concept of molecular replacement. This method can be applied to proteins with similar structure for example, high sequence homologies, mutant proteins, proteins after chemical modification or substrate-binding protein. 

Left side of Fig. 4 shows a ribbon model of the catalytic (C-) subunit of the mammalian cAMP-dependent protein kinase. This was the first protein kinase whose structure was determined [35]. Figure 4 includes also a ribbon model of the peptide substrate, and ATP (stick representation) with two manganese ions (CPK representation). All kinetic evidence is consistent with a preferred ordered mechanism of catalysis with ATP binding proceeding substrate binding. 

Although FeMo-cofactor is clearly knpHcated in substrate reduction cataly2ed by the Mo-nitrogenase, efforts to reduce substrates using the isolated FeMo-cofactor have been mosdy equivocal. Thus the FeMo-cofactor s polypeptide environment must play a critical role in substrate binding and reduction. Also, the different spectroscopic features of protein-bound vs isolated FeMo-cofactor clearly indicate a role for the polypeptide in electronically fine-tuning the substrate-reduction site. Site-directed amino acid substitution studies have been used to probe the possible effects of FeMo-cofactor s polypeptide environment on substrate reduction (163—169). Catalytic and spectroscopic consequences of such substitutions should provide information concerning the specific functions of individual amino acids located within the FeMo-cofactor environment (95,122,149). 

The theory predicts that such proteins are built up of several subunits which are symmetrically arranged and that the two states differ by the arrangements of the subunits and the number of bonds between them. In one state the subunits are constrained by strong bonds that would resist the structural changes needed for substrate binding, and this state would consequently bind substrates weakly they called it the tense or T state. In the other state, called the R state, these constraints are relaxed. 

This idea also helps to explain some of the mystery surrounding the enormous catalytic power of enzymes In enzyme catalysis, precise orientation of catalytic residues comprising the active site is necessary for the reaction to occur substrate binding induces this precise orientation by the changes it causes in the protein s conformation. 

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