Intramolecular catalysis of a proline isomerization reaction in the folding of dihydrofolate reductase.
The cis/trans isomerization of the peptide bond preceding proline residues in proteins can limit the rate at which a protein folds to its native conformation. Mutagenic analyses of dihydrofolate reductase (DHFR) from Escherichia coli show that this isomerization reaction can be intramolecularly catalyzed by a side chain from an amino acid which is distant in sequence but adjacent in the native conformation. The guanidinium NH2 nitrogen of Arg 44 forms one hydrogen bond to the imide nitrogen and a second to the carbonyl oxygen of Pro 66 in wild-type DHFR. Replacement of Arg 44 with Leu results in a change of the nature of the two slow steps in refolding from being limited by the acquisition of secondary and/or tertiary structure to being limited by isomerization. The simultaneous replacement of Pro 66 with Ala (i.e., the Leu 44/Ala 66 double mutant) eliminates this isomerization reaction and once again makes protein folding the limiting process. Apparently, one or both of the hydrogen bonds between Arg 44 and Pro 66 accelerate the isomerization of the Gln 65-Pro 66 peptide bond. The replacement of Arg 44 with Leu affects the kinetics of the slow folding reactions in a fashion which indicates that the crucial hydrogen bonds form in the transition states for the rate-limiting steps in folding.
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Department of Chemistry, Albright College, Reading, Pennsylvania 19620.
Spencer
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Rosenstein
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Matthews
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D001120:Arginine / Q000737:chemistry; D001483:Base Sequence; D002384:Catalysis; D004926:Escherichia coli / Q000201:enzymology; D006860:Hydrogen Bonding; D007536:Isomerism; D008969:Molecular Sequence Data; D015394:Molecular Structure; D016297:Mutagenesis, Site-Directed; D009841:Oligonucleotides / Q000737:chemistry; D011392:Proline / Q000737:chemistry*; D011487:Protein Conformation; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry* / Q000235:genetics; D014961:X-Ray Diffraction
D009841:Oligonucleotides; D001120:Arginine; D011392:Proline; D013762:Tetrahydrofolate Dehydrogenase