STRUCTURE AND FUNCTION OF ALTERNATIVE PROTON-RELAY MUTANTS OF DIHYDROFOLATE-REDUCTASE

被引:23
作者
DAVID, CL
HOWELL, EE
FARNUM, MF
VILLAFRANCA, JE
OATLEY, SJ
KRAUT, J
机构
[1] UNIV CALIF SAN DIEGO,DEPT CHEM,LA JOLLA,CA 92093
[2] UNIV TENNESSEE,DEPT BIOCHEM,KNOXVILLE,TN 37996
[3] AGOURON PHARMACEUT INC,SAN DIEGO,CA 92121
关键词
D O I
10.1021/bi00155a038
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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 pK(a) 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.
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页码:9813 / 9822
页数:10
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