Specificity of the Valyl Ribonucleic Acid Synthetase from Escherichia coli in the Binding of Valine Analogues

A series of modified trp operator sequences has been prepared by the incorporation of seven different base analogues. Four of the analogues allow the site-specific deletion of functional groups present on the dA-dT and dT-dA base pairs at positions -4/+4 and -5/+5 in the trp operator. The remaining three analogues permit the incorporation of structural analogues of the native dA-dT or dG-dC base pairs. The duplex operator sequences all exhibit Tm values well above ambient temperature (48-70 degrees C), and these values generally correlate very well with the number of interstrand hydrogen bonds present. The affinity between the trp repressor and 14 modified operator sequences was examined using a recently developed alkaline phosphatase protection assay. The results from the analogue sequences used in this study suggest that the structure of the dA-dT or dT-dA base pairs at positions -4/+4 and -5/+5, respectively, has relatively little effect upon the solution binding by the trp repressor, but the protein is very sensitive to the orientation of the amino and carbonyl functional groups at the -4/+4 positions, which are involved in the formation of an interbase hydrogen bond present in the major groove. (The term structure in this case refers to the hydrogen bonding structure of the base pairs. We recognize that the introduction of conservative functional group deletions or reversals may affect other structural criteria such as hydration.) The deletion of individual functional groups from the operator sequence suggests that the carbonyl at dT+4 is critical for formation of the high-affinity sequence-specific complex. Additionally, the thymine methyl group at dT+4 and the N7 nitrogen of dA+5 appear to be critical contacts necessary for high-affinity binding by the repressor. The thymine carbonyl and the adenine N7 nitrogen are each responsible for approximately -1.5 kcal/mol of apparent free energy of binding. The thymine methyl provides a somewhat smaller contribution of -0.7 kcal/mol. Deletion of either of the adenine amino groups at dA-4 or dA+5 results in a sequence that binds to the repressor with a higher affinity than observed with the native sequence; this can be explained in that the functional groups lost are not critical for binding, and the resulting increased flexibility of the operator, or the creation of a more hydrophobic surface at these sites, enhances van der Waals contacts between the protein and the nucleic acid.

Section: Discussionmentioning

confidence: 99%

Interactions between the trp repressor and its operator sequence as studied by base analog substitution

Mazzarelli¹,

Rajur²,

Iadarola³

et al. 1992

“…The energy contributed by a substrate CH3 group has been analyzed for several proteins and varies between 6.5 kJ mol"1 for chymotrypsin (Dorovska et al, 1972) and 14 kJ mol"1 for various aminoacyl-tRNA synthetases (Loftfield & Eigner, 1966;Owens & Bell, 1970;Fersht & Dingwall, 1979;Fersht et al, 1980). The values of-14 kJ mol"1 kJ mol"1 seen with tRNA synthetases indicate an extremely well formed CH3 binding hole that makes a very tight interaction with the methyl group.…”

Section: Discussionmentioning

confidence: 99%

Interaction of the EcoRV restriction endonuclease with the deoxyadenosine and thymidine bases in its recognition hexamer d(GATATC)

Newman¹,

Williams²,

Cosstick³

et al. 1990

A set of dA and T analogues suitable for the study of protein DNA interactions have been incorporated into the central d(ATAT) sequence within d(GACGATATCGTC). The individual analogues have one potential protein contact (either a hydrogen-bonding group or a CH3 group capable of a van der Waals interaction) deleted. In general, the modified bases do not perturb the overall structure of the dodecamer, enabling results obtained to be simply interpreted in terms of loss of protein DNA contacts. We have used the modified oligodeoxynucleotide set to study the recognition of DNA by the EcoRV restriction endonuclease [recognition sequence d(GATATC)]. The kcat and Km values for the set have been determined, and a comparison with results seen with the parent oligodeoxynucleotide (containing no modified bases) has been carried out. Three classes of results are seen. First, some analogues lead to no change in kinetic parameters, meaning no enzyme contact at the altered site. Second (this is seen for most of the modified oligodeoxynucleotides), a drop in the kcat/Km ratio relative to the parent is observed. This comes mainly from a decrease in kcat, implying that the endonuclease uses the interaction under study to lower the transition-state barrier rather than to bind the substrate. Analyses of these results show that the drop in kcat/Km is what would be expected for the simple loss of a hydrogen bond or a CH3 contact between the enzyme and the oligodeoxynucleotide. This implies a contact of these types at these sites.(ABSTRACT TRUNCATED AT 250 WORDS)

“…The -amino group appears to be a major binding locus for a number of aa-tRNA synthetases and is absolutely essential for substrate activity (Calendar and Berg, 1966;Papas and Mehler, 1970;Conway et at., 1962;Owens and Bell, 1970). Since, with the exception of proline, the primary amino group is found in all of the ligase substrates, it cannot in any obvious manner serve to select the proper amino acid; however, in view of its rudimentary role in binding and the sensitivity of binding toward structural modifications, the primary amino group must certainly be one of the common recognition sites for this group of enzymes.…”

Section: Discussionmentioning

confidence: 99%

“…In the present work, competitive inhibitors are used to analyze the topography and localized environment of the L-phenylalanine binding site of PRS1 from E. coli B. Comparisons of the results obtained with similar studies of other activating enzymes (Papas and Mehler, 1970;Owens and Bell, 1970;Calendar and Berg, 1966) permit speculation on features which may be relevant to general questions of catalysis and specificity.…”

mentioning

confidence: 96%

“…A number of amino acid analogs are able to meet the requirements for the binding and activation steps, and in some instances are esterified to tRNA (see, for example, Owens and Bell, 1970, Calendar andBerg, 1966, Conway et al, 1962, the a hydrogen of L-phenylalanine; groups larger than hydrogen perturb other binding loci and lead to large losses in affinity. The presence of the -carboxyl group is not necessary for optimal binding, and substitution by hydrophobic groups leads to substantial increases in affinity for the enzyme.…”

mentioning

confidence: 99%

See 1 more Smart Citation

Phenylalanyl transfer ribonucleic acid synthetase from Escherichia coli. Analysis of the phenylalanine binding site

Santi¹,

Danenberg²

1971

Using structural analogs of L-phenylalanine, a model has been constructed which describes the amino acid binding site of Phe-tRNA synthetase from Escherichia coli B. The area of the enzyme which complexes the benzene ring of L-phenylalanine represents the primary site of recognition and one of the two major binding loci. This region is best described as a hydrophobic pocket with a stringent steric