The role of tryptophan-48 in catalysis and binding of inhibitors of Plasmodium falciparum dihydrofolate reductase.
Dihydrofolate reductases (DHFRs) from Plasmodium falciparum (Pf) and various species of both prokaryotic and eukaryotic organisms have a conserved tryptophan (Trp) at position 48 in the active site. The role in catalysis and binding of inhibitors of the conserved Trp48 of PfDHFR has been analysed by site-specific mutagenesis, enzyme kinetics and use of a bacterial surrogate system. All 19 mutant enzymes showed undetectable or very low specific activities, with the highest value of k(cat)/K(m) from the Tyr48 (W48Y) mutant (0.12 versus 11.94M(-1)s(-1)), of about 1% of the wild-type enzyme. The inhibition constants for pyrimethamine, cycloguanil and WR99210 of the W48Y mutants are 2.5-5.3 times those of the wild-type enzyme. All mutants, except W48Y, failed to support the growth of Escherichia coli transformed with the parasite gene in the presence of trimethoprim, indicating the loss of functional activity of the parasite enzyme. Hence, Trp48 plays a crucial role in catalysis and inhibitor binding of PfDHFR. Interestingly, W48Y with an additional mutation at Asn188Tyr (N188Y) was found to promote bacterial growth and yielded a higher amount of purified enzyme. However, the kinetic parameters of the purified W48Y+N188Y enzyme were comparable with W48Y and the binding affinities for DHFR inhibitors were also similar to the wild-type enzyme. Due to its conserved nature, Trp48 of PfDHFR is a potential site for interaction with antimalarial inhibitors which would not be compromised by its mutations.
Kamchonwongpaisan
Sumalee
S
National Center for Genetic Engineering and Biotechnology, National Science and Technology Development Agency, Pathumthani, Thailand.
Vanichtanankul
Jarunee
J
Taweechai
Supannee
S
Chitnumsub
Penchit
P
Yuthavong
Yongyuth
Y
D000818:Animals; D002384:Catalysis; D016054:DNA, Protozoan / Q000737:chemistry / Q000235:genetics; D004795:Enzyme Stability; D004926:Escherichia coli / Q000254:growth & development; D005493:Folic Acid Antagonists / Q000378:metabolism / Q000494:pharmacology; D005816:Genetic Complementation Test; D007700:Kinetics; D016254:Mutagenesis, Insertional; D010963:Plasmodium falciparum / Q000201:enzymology* / Q000235:genetics / Q000378:metabolism; D002727:Proguanil / Q000494:pharmacology; D011739:Pyrimethamine / Q000494:pharmacology; D013762:Tetrahydrofolate Dehydrogenase / Q000235:genetics / Q000378:metabolism*; D014227:Triazines / Q000494:pharmacology; D014364:Tryptophan / Q000235:genetics / Q000378:metabolism*
D016054:DNA, Protozoan; D005493:Folic Acid Antagonists; D014227:Triazines; C026009:cycloguanil; C006201:BRL 6231; D014364:Tryptophan; D013762:Tetrahydrofolate Dehydrogenase; D002727:Proguanil; D011739:Pyrimethamine