Nucleotide-dependent conformational change governs specificity and analog discrimination by HIV reverse transcriptase.
Single turnover studies on HIV reverse transcriptase suggest that nucleoside analogs bind more tightly to the enzyme than normal substrates, contrary to rational structural predictions. Here we resolve these controversies by monitoring the kinetics of nucleotide-induced changes in enzyme structure. We show that the specificity constant for incorporation of a normal nucleotide (dCTP) is determined solely by the rate of binding (including isomerization) because isomerization to the closed complex commits the substrate to react. In contrast, a nucleoside analog (3TC-TP, triphosphate form of lamivudine) is incorporated slowly, allowing the conformational change to come to equilibrium and revealing tight nucleotide binding. Our data reconcile previously conflicting reports suggesting that nucleotide analogs bind tighter than normal nucleotides. Rather, dCTP and 3TC-TP bind with nearly equal affinities, but the binding of dCTP never reaches equilibrium. Discrimination against 3TC-TP is based on the slower rate of incorporation due to misalignment of the substrate and/or catalytic residues.
Kellinger
Matthew W
MW
Institute for Cellular and Molecular Biology, University of Texas, Austin, TX 78712, USA.
Johnson
Kenneth A
KA
D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D003570:Cytidine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism; D017931:DNA Primers / Q000235:genetics; D054306:Dideoxynucleotides / Q000378:metabolism*; D005470:Fluorometry; D054303:HIV Reverse Transcriptase / Q000235:genetics* / Q000378:metabolism*; D007700:Kinetics; D019259:Lamivudine / Q000031:analogs & derivatives* / Q000378:metabolism; D008956:Models, Chemical; D016296:Mutagenesis; D011485:Protein Binding / Q000235:genetics; D011487:Protein Conformation; D013379:Substrate Specificity / Q000235:genetics
D017931:DNA Primers; D054306:Dideoxynucleotides; C417268:lamivudine triphosphate; D019259:Lamivudine; D003570:Cytidine Triphosphate; D054303:HIV Reverse Transcriptase
Science. 1998 Nov 27;282(5394):1669-75
9831551
Biochemistry. 2004 Nov 16;43(45):14317-24
15533035
Science. 1995 Feb 17;267(5200):988-93
7532321
Antimicrob Agents Chemother. 2000 Jan;44(1):217-21
10602755
Mol Cell. 2005 Aug 5;19(3):345-55
16061181
J Mol Biol. 1998 Dec 11;284(4):1095-111
9837729
Proc Natl Acad Sci U S A. 1995 Aug 15;92(17):8046-9
7544013
Nucleic Acids Res. 2007;35(20):6973-83
17940100
Annu Rev Biochem. 2006;75:519-41
16756501
J Biol Chem. 1998 Oct 16;273(42):27250-8
9765248
Chem Biol. 1999 May;6(5):R137-46
10322129
FEBS Lett. 1991 Oct 7;291(1):1-5
1718777
Proc Natl Acad Sci U S A. 2007 Jul 31;104(31):12610-5
17640918
Annu Rev Biochem. 1993;62:685-713
7688945
J Biol Chem. 1992 Dec 25;267(36):25988-97
1281479
Biochemistry. 2008 Mar 18;47(11):3317-21
18298083
Biochemistry. 2006 Aug 15;45(32):9675-87
16893169
Biochemistry. 1999 Jan 5;38(1):55-63
9890882
Biochemistry. 1997 Nov 18;36(46):14064-70
9369478
Biochemistry. 2002 Apr 23;41(16):5150-62
11955063
Anal Biochem. 2008 Jan 1;372(1):125-7
17905190
Biochemistry. 2002 Aug 27;41(34):10571-6
12186540
FASEB J. 1999 Sep;13(12):1511-7
10463941
Anal Biochem. 2009 Jan 1;384(1):136-44
18834851