The mechanism of action of dihydrofolate reductase revealed by nonresonance Raman difference spectroscopy.
Item
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Title
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The mechanism of action of dihydrofolate reductase revealed by nonresonance Raman difference spectroscopy.
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Identifier
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AAI9510645
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identifier
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9510645
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Creator
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Chen, Yong-Qing.
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Contributor
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Adviser: Robert H. Callender
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Date
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1994
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Language
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English
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Publisher
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City University of New York.
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Subject
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Biophysics, General | Chemistry, Biochemistry
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Abstract
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We have studied the interactions of dihydrofolate reductase (DHFR) with NADP coenzymes, folate substrates and inhibitors using nonresonance difference Raman spectroscopy.;A 35 cm{dollar}\sp{lcub}-1{rcub}{dollar} upshift observed in the rocking motion the carboxamide {dollar}-{dollar}NH{dollar}\sb2{dollar} group NADPH upon binding E. coli DHFR (ecDHFR) indicates that this group forms strong hydrogen bonds in the active site. These interaction appear to be responsible for holding the nicotinamide ring in close contact with protein groups, causing 44 cm{dollar}\sp{lcub}-1{rcub}{dollar} upshift of the C4-D stretch frequency of NADPD{dollar}\sb{lcub}\rm B{rcub}{dollar} bound to the binary complex. Two C4-D stretch bands were observed for NADPD{dollar}\sb{lcub}\rm A{rcub}{dollar} or NADPD{dollar}\sb{lcub}\rm B{rcub}{dollar} bound the ternary complex with methotrexate (MTX), suggesting two environments for the nicotinamide ring.;We have observed three forms of biopterin or folate in the ecDHFR ternary complex with NADP{dollar}\sp+{dollar}, a slightly polarized keto form with C4=0 stretch at 1693 cm{dollar}\sp{lcub}-1{rcub}{dollar}, a highly polarized keto form with C4{dollar}\underline{lcub}\cdot\cdot{rcub}{dollar} O stretch at 1653 cm{dollar}\sp{lcub}-1{rcub}{dollar}, and an enolate form with C4-O{dollar}\sp-{dollar} stretch at 1341 cm{dollar}\sp{lcub}-1{rcub}{dollar}.;We have measured the Raman difference spectrum between ecDHFR and the Asp-27 to serine mutant (D27S), and found no evidence for Asp-27 having an elevated pK{dollar}\sb{lcub}\rm a{rcub}{dollar} of 6.5. Instead, we have found the pK{dollar}\sb{lcub}\rm a{rcub}{dollar} of N5 of dihydrofolate in the ecDHFR ternary complex with NADP{dollar}\sp+{dollar} is elevated to 6.5 from 2.6 in solution. This value is found to be no lower than 5 in human DHFR ternary complex with 1,4,5,6-tetrahydronicotinamide adenine dinucleotide phosphate (H{dollar}\sb2{dollar}NADPH). In contrast, the pK{dollar}\sb{lcub}\rm a{rcub}{dollar} of N5 is less than 4.0 in the ecDHFR ternary complex with H{dollar}\sb2{dollar}NADPH, the D27S ternary complex with NADP{dollar}\sp+{dollar}, or the binary complex with either ecDHFR or human DHFR.;We have also found that MTX can be reduced to 5,8-dihydro-MTX in the ecDHFR ternary complex with NADPH. Irradiation of laser light with wavelength below 568.2 nm will greatly increase the reaction equilibrium constant. The reduced MTX is slowly oxidized back to MTX, probably by reacting with protein side chains or oxygen in solution.
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Type
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dissertation
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Source
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PQT Legacy CUNY.xlsx
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degree
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Ph.D.
