Structure and function of alternative proton-relay mutants of dihydrofolate reductase.
Using site-specific mutagenesis, we have constructed two mutants of Escherichia coli dihydrofolate reductase (ecDHFR) to investigate further the function of a weakly acidic side chain at position 27 in substrate protonation: Asp27-->Glu (D27E) and Asp27-->Cys (D27C). The crystal structure of D27E ecDHFR in a binary complex with methotrexate shows that the side-chain oxygen atoms of Glu27 are in almost precisely the same location as those of Asp27 in the wild-type enzyme. Kinetic evidence indicates that Glu27 can indeed function efficiently in the proton relay to dihydrofolate. Even though vertebrate DHFRs all have a glutamic acid at the structurally equivalent position, the kinetic properties of Glu27 ecDHFR more closely resemble those of wild-type bacterial DHFRs than of vertebrate DHFRs. The D27C mutation produced an enzyme still capable of relaying a proton to dihydrofolate, but with the intrinsic pKa in its pH-activity profiles shifted upward to values characteristic of the more basic thiolate group. The crystal structure of the binary complex with methotrexate reveals two unexpected features: (1) the Cys27 sulfhydryl group does not point toward the pteridine-binding site, but the side chain of this residue is instead rotated 120 degrees to interact with a tyrosine side chain projecting from a neighboring beta-strand; (2) a bound ethanol molecule occupies a cavity adjacent to methotrexate. Ethanol is a component of the crystallization medium.
Department of Chemistry, University of California, San Diego, La Jolla 92093.
C L
CL
David
E E
EE
Howell
M F
MF
Farnum
J E
JE
Villafranca
S J
SJ
Oatley
J
J
Kraut
D001216:Asparagine / Q000378:metabolism; D001665:Binding Sites; D003461:Crystallography; D004926:Escherichia coli / Q000201:enzymology; D005971:Glutamates / Q000378:metabolism; D018698:Glutamic Acid; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008727:Methotrexate / Q000378:metabolism; D016297:Mutagenesis, Site-Directed; D011522:Protons; D011621:Pteridines / Q000378:metabolism; D013056:Spectrophotometry, Ultraviolet; D013762:Tetrahydrofolate Dehydrogenase / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*
D005971:Glutamates; D011522:Protons; D011621:Pteridines; D018698:Glutamic Acid; D001216:Asparagine; D013762:Tetrahydrofolate Dehydrogenase; D008727:Methotrexate