Comparative analysis of native and cysteine-deficient HIV-1 reverse transcriptase.
To study the subunit structure and the active site of human immunodeficiency virus reverse transcriptase (RT), the enzyme was expressed in E. coli and purified to homogeneity in large quantities. The recombinant enzyme consists of two major polypeptides of 66,000 and 53,000 Da in equimolar amounts and a minor species of 51,000 Da. Amino acid sequence analysis of the recombinant proteins revealed that the amino termini of the two major subunits are identical to that of the virion-derived enzyme. The two cysteinyl residues at positions 38 and 280 in the RT amino acid sequence were replaced by alanine in an attempt to elucidate the role of the sulfhydryl groups in RT enzyme activities, heterodimer formation, and intrasubunit linkage. The results reported here show that the two cysteinyls are dispensable and their absence in the amino acid sequence of the reverse transcriptase does not affect DNA polymerase or ribonuclease H enzyme activities or the formation of heterodimer structures. Furthermore, inhibitors of polymerase activity such as 3-azidothymidine triphosphate, dideoxythymidine triphosphate, and tetrahydroimidazo[4,5,1-JK][1,4]benzodiazepens (1H)-one are equally effective on the mutant containing no cysteinyl residues and the wild-type enzyme.
Fischer
M
M
Biotechnology General, Israel Ltd, Kiryat Weizmann, Rehovot.
Lifshitz
R
R
Katz
T
T
Liefer
I
I
Ben-Artzi
H
H
Gorecki
M
M
Panet
A
A
Zeelon
E
E
D001483:Base Sequence; D003545:Cysteine / Q000502:physiology*; D054303:HIV Reverse Transcriptase; D008969:Molecular Sequence Data; D008970:Molecular Weight; D016297:Mutagenesis, Site-Directed; D011487:Protein Conformation; D012194:RNA-Directed DNA Polymerase / Q000737:chemistry* / Q000235:genetics; D011994:Recombinant Proteins / Q000737:chemistry / Q000235:genetics / Q000378:metabolism; D016914:Ribonuclease H / Q000032:analysis; D013329:Structure-Activity Relationship
D011994:Recombinant Proteins; D054303:HIV Reverse Transcriptase; D012194:RNA-Directed DNA Polymerase; D016914:Ribonuclease H; D003545:Cysteine