Biomaterial Database

The effects of cysteine mutations on the catalytic activities of the reverse transcriptase of human immunodeficiency virus type-1. 1992

S Loya, and R Tal, and S H Hughes, and A Hizi

Department of Cell Biology and Histology, Sackler School of Medicine, Tel Aviv University, Israel.

The reverse transcriptase (RT) of the human immunodeficiency virus type 1 (HIV-1) has only 2 cysteine residues at positions 38 and 280. In order to investigate the role of these cysteines in the structure and function of the enzyme, we have previously modified each of the cysteines to serines employing site-directed mutagenesis. Two of the mutant forms of HIV-1 RT, the single mutant of cysteine 280 and a double mutant with both cysteines modified, were purified. In the present study we have compared the catalytic properties of the DNA-polymerizing and the ribonuclease H (RNase H) functions of the two mutant RTs to those of the native enzyme. The results indicate that the single mutant RT closely resembles the wild type enzyme in almost all the catalytic functions tested. The double cysteine mutant RT, on the other hand, exhibits several unique features. First, the specific activities of the RNA- and DNA-directed DNA synthesis are significantly lower than the corresponding activities of the other two enzymes. This probably results from the lower Vmax values exhibited by the double mutant RT, since the Km values calculated for all enzymes were similar. Second, the most outstanding differences are associated with the RNase H activity of the double mutant RT. The specific activity of RNase H is about 4-fold higher than the wild type and the single mutant RTs. Furthermore, the heat stability of the RNase H function of the double mutated RT is at least 15-fold higher than that of the other two RTs. The substantial resistance to heat denaturation is apparent only for the RNase H activity, since the DNA polymerizing function of the double mutant RT is as sensitive to heat denaturation as the other two proteins.

UI	MeSH Term	Description	Entries
D007700	Kinetics	The rate dynamics in chemical or physical systems.
D011089	Polydeoxyribonucleotides	A group of 13 or more deoxyribonucleotides in which the phosphate residues of each deoxyribonucleotide act as bridges in forming diester linkages between the deoxyribose moieties.	Polydeoxyribonucleotide
D011994	Recombinant Proteins	Proteins prepared by recombinant DNA technology.	Biosynthetic Protein,Biosynthetic Proteins,DNA Recombinant Proteins,Recombinant Protein,Proteins, Biosynthetic,Proteins, Recombinant DNA,DNA Proteins, Recombinant,Protein, Biosynthetic,Protein, Recombinant,Proteins, DNA Recombinant,Proteins, Recombinant,Recombinant DNA Proteins,Recombinant Proteins, DNA
D003545	Cysteine	A thiol-containing non-essential amino acid that is oxidized to form CYSTINE.	Cysteine Hydrochloride,Half-Cystine,L-Cysteine,Zinc Cysteinate,Half Cystine,L Cysteine
D004795	Enzyme Stability	The extent to which an enzyme retains its structural conformation or its activity when subjected to storage, isolation, and purification or various other physical or chemical manipulations, including proteolytic enzymes and heat.	Enzyme Stabilities,Stabilities, Enzyme,Stability, Enzyme
D004926	Escherichia coli	A species of gram-negative, facultatively anaerobic, rod-shaped bacteria (GRAM-NEGATIVE FACULTATIVELY ANAEROBIC RODS) commonly found in the lower part of the intestine of warm-blooded animals. It is usually nonpathogenic, but some strains are known to produce DIARRHEA and pyogenic infections. Pathogenic strains (virotypes) are classified by their specific pathogenic mechanisms such as toxins (ENTEROTOXIGENIC ESCHERICHIA COLI), etc.	Alkalescens-Dispar Group,Bacillus coli,Bacterium coli,Bacterium coli commune,Diffusely Adherent Escherichia coli,E coli,EAggEC,Enteroaggregative Escherichia coli,Enterococcus coli,Diffusely Adherent E. coli,Enteroaggregative E. coli,Enteroinvasive E. coli,Enteroinvasive Escherichia coli
D000595	Amino Acid Sequence	The order of amino acids as they occur in a polypeptide chain. This is referred to as the primary structure of proteins. It is of fundamental importance in determining PROTEIN CONFORMATION.	Protein Structure, Primary,Amino Acid Sequences,Sequence, Amino Acid,Sequences, Amino Acid,Primary Protein Structure,Primary Protein Structures,Protein Structures, Primary,Structure, Primary Protein,Structures, Primary Protein
D012194	RNA-Directed DNA Polymerase	An enzyme that synthesizes DNA on an RNA template. It is encoded by the pol gene of retroviruses and by certain retrovirus-like elements. EC 2.7.7.49.	DNA Polymerase, RNA-Directed,RNA-Dependent DNA Polymerase,Reverse Transcriptase,RNA Transcriptase,Revertase,DNA Polymerase, RNA Directed,DNA Polymerase, RNA-Dependent,RNA Dependent DNA Polymerase,RNA Directed DNA Polymerase
D013379	Substrate Specificity	A characteristic feature of enzyme activity in relation to the kind of substrate on which the enzyme or catalytic molecule reacts.	Specificities, Substrate,Specificity, Substrate,Substrate Specificities
D013816	Thermodynamics	A rigorously mathematical analysis of energy relationships (heat, work, temperature, and equilibrium). It describes systems whose states are determined by thermal parameters, such as temperature, in addition to mechanical and electromagnetic parameters. (From Hawley's Condensed Chemical Dictionary, 12th ed)	Thermodynamic