Polypeptide complexes, substitution

One specific question which I would like to address to Dr. Margerum concerns the very interesting series of substitution reactions for the polypeptide complexes that are of similar stability and yet exhibit remarkably different substitution rates. What would you say about the implications of that observation for the formation processes of the copper polypeptide complexes ... 

Fig. 17. Structure of a macromolecular polyornithine Gd-D03A complex, note that the 84 unsubstituted and the 30 substituted residues are randomly distributed on the polypeptide chain...

Site-directed mutagenesis, by which one amino acid in a protein is substituted for another, is an established and invaluable tool in protein chemistry. However, the size and complexity of proteins often precludes unambiguous interpretation of the results of such experiments. This is due to the non-uniform distribution of structural information within a protein sequence - the fact that some residues are tolerant to substitution, whereas others cannot be replaced without deleterious consequences - and to the many energetically similar conformational states available to any polypeptide. These factors, and the extreme sensitivity of catalytic activity to seemingly modest structural perturbation, make characterization and (re)design of enzymes in the laboratory difficult. 

Meisenheimer complex. On the addition of a protic acid, the complex may fragment with the elimination of either the ethoxide or methoxide anions. If the ethoxide anion is eliminated this results in an overall substitution of an ethoxy group for methoxy group at the ipso position of the aromatic ring. Another example would involve the use of 2,4-dinitrofluorobenzene, which is used to form the derivative of the amino terminal of polypeptides. 

FIGURE 8.3 A hypothetical series of isomorphous heavy atom derivatives for a crystalline macromolecule, represented here by the polypeptide backbone of rubredoxin. (a) The apo-protein, stripped of its metal ion, provides native structure factors />, shown in vector and waveform on the right (b) the protein with its naturally bound iron atom and FHi, the first derivative structure factor (c) the protein with its iron plus an attached mercury atom, and the resultant structure factor Fm from the double derivative (d) a second multiply substituted derivative formed by attachment of a gold atom to the protein-iron complex. This last derivative is only marginally useful, however, since the reaction with gold also produces a modification in the tertiary structure of the protein (denoted by an arrow). Since this non-isomorphism is equivalent to introducing a nonnative structure factor contribution, the observed F s cannot be properly accounted for, and an erroneous heavy atom contribution / results. This final derivative will yield an inaccurate phase estimate 0v for the native protein. 

Stable structures such as the naturally occurring ferric porphyrin complexes or porphyrins substituted with copper, cobalt, silver or vanadyl probe the active site of heme-containing enzyme [227]. Complexes of copper not associated with heme are also common. They are frequently formed at an amino terminus because the amino group provides a good primary amine donor atom. Two or three amino acid residues beginning at the N-terminus are often flexible until a more rigid portion of the polypeptide, such as the a helix, is encountered. A peptide nitrogen is available to... 

Figure 4. Proposed plastoquinine (QB) and herbicide binding site on the 32 kDalton D-1 polypeptide of photosystem II. The quinone is bound through an iron-complexed histidine residue (his 215) and hydrogen bonding to ser 264. Further interactions occur with arg 269 and phe 255 lying above and below the binding site. Amino acid substitutions in herbicide-tolerant mutants have been identified at the residues numbered 219. 255, 264 and 275. Reproduced with permission from Ref. 57. Copyright 1986 Verlag der Zeitschrift fur Naturforschung.

Active sites in de novo designed polypeptides are, as a rule, built from surface exposed residues, even if the polypeptide is folded. The design from scratch of proteins or polypeptides that fold to form cavities is still in its infancy and systematic variations are, by necessity, difficult in complex structures because the structures may change with amino acid substitutions. Rate enhancements of three to four orders of magnitude have been reported several times in designed catalysts [4-9] but those of typical enzymes are unrealistic in solvent exposed catalytic sites. The number of functional groups that can interact with substrates, intermediates and transition states is limited and the many degrees of freedom of the active site residues reduce the catalytic efficiency for entropic reasons. However, incorporation of an active site developed in a surface catalyst into a constrained hydrophobic pocket... 

Two structures of LDH have been determined, that of apo-LDH and that of the abortive ternary complex LDHrNAD-pyruvate. In both these structures the tetrameric LDH molecule has strict 222 symmetry with all four subunits identical. In the tetrameric holo-GAPDH, on the other hand, all four subunits are crystallographically independent and might then have subtle structural differences. In the crystal structure determination of s-MDH (a dimer) there were two crystallographically independent polypeptide chains. These were differentially substituted by the... 

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