Comparative study of binase and barnase: experience in chimeric ribonucleases

Schulga, Alexey; Kurbanov, F. T.; Kirpichnikov, Mikhaǐl P.; Protasevich, I.I.; Lobachov, Vladimir M.; Ranjbar, Bijan; Chekhov, V. O.; Polyakov, K. M.; Engelborghs, Yves; Makarov, Alexander А.

doi:10.1093/protein/11.9.775

Cited by 48 publications

(46 citation statements)

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“…This affinity compares favorably with the K M of binase for oligoribonucleotides (between 100 and 400 M for different substrates; ref. 31), suggesting that a functionally relevant binding is being monitored. The extent of the shift changes suggests that major structural rearrangements occur in the protein.…”

Section: Resultsmentioning

confidence: 99%

Functional dynamics in the active site of the ribonuclease binase

Wang

Pang

Holder

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

114

134

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Binase, a member of a family of microbial guanyl-specific ribonucleases, catalyzes the endonucleotic cleavage of single-stranded RNA. It shares 82% amino acid identity with the well-studied protein barnase. We used NMR spectroscopy to study the millisecond dynamics of this small enzyme, using several methods including the measurement of residual dipolar couplings in solution. Our data show that the active site of binase is flanked by loops that are flexible at the 300-s time scale. One of the catalytic residues, His-101, is located on such a flexible loop. In contrast, the other catalytic residue, Glu-72, is located on a ␤-sheet, and is static. The residues Phe-55, part of the guanine base recognition site, and Tyr-102, stabilizing the base, are the most dynamic. Our findings suggest that binase possesses an active site that has a well-defined bottom, but which has sides that are flexible to facilitate substrate access͞egress, and to deliver one of the catalytic residues. The motion in these loops does not change on complexation with the inhibitor d(CGAG) and compares well with the maximum kcat (1,500 s ؊1 ) of these ribonucleases. This observation indicates that the NMR-measured loop motions reflect the opening necessary for product release, which is apparently rate limiting for the overall turnover. It has been inferred from the inspection of static structures that many enzymes sequester substrate or inhibitor from solution and release product by dynamically opening and closing their active site areas (1). Recently, NMR spectroscopy has become an important experimental method to obtain dynamic characterization for individual sites in proteins and nucleic acids (2). Here, we use several complementary NMR techniques-measurement of conformational exchange line broadening, measurement of residual dipolar couplings, and measurement of amide proton exchange-to characterize slower motions in the ribonuclease binase. This protein is a member of a family of homologous guanyl-specific ribonucleases that catalyze the cleavage of singlestranded RNA via a two-step mechanism involving transesterification with formation of a 2Ј,3Ј cyclic phosphate intermediate followed by hydrolysis to yield 3Ј-phosphate. The residues His-101 and Glu-72 are involved in the acid-base catalysis of both of these steps (see Fig. 3 A and B and Refs. 3 and 4). Binase is expressed by Bacillus intermedius and is a 109-aa protein that differs in 17 residues and 1 deletion from the well-studied ribonuclease barnase (4). The substitutions are outside the active center and are mainly on the surface of the protein. MethodsConformational Exchange Broadening: Theory. The conformational exchange linebroadening R ex contributes in the fast͞intermedi-ate limit to the 15 N transverse relaxation rate R 2 as R 2 ϭ R 2 0 ϩ R ex , where R 2 0 is the natural relaxation rate. In the free precession case, the broadening R ex caused by conformational exchange at a rate ex Ϫ1 between two sites A and B with fractions f A and f B , and a radial chemical shift difference ...

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Section: Resultsmentioning

confidence: 99%

Functional dynamics in the active site of the ribonuclease binase

Wang

Pang

Holder

et al. 2001

Proc. Natl. Acad. Sci. U.S.A.

114

134

View full text Add to dashboard Cite

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“…The enzyme was purified as described earlier. 41 Endotoxins content in binase preparations, determined by the Limulus amoebocyte lysate test (LAL) (Charles River Endosafe), was less than 5 EU/mg. Binase was assayed for catalytic activity using poly(I) as substrate.…”

Section: Methodsmentioning

confidence: 99%

Ribonuclease binase inhibits primary tumor growth and metastases via apoptosis induction in tumor cells

et al. 2013

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“…The enzyme purification was described earlier. 28 Binase was assayed for catalytic activity using both synthetic substrates 28 and yeast RNA. 29 Imatinib (Gleevec, Novartis, Basel, Switzerland) was dissolved in water at 10 mM and used at final concentrations from 0.1 to 1.0 µM.…”

Section: Methodsmentioning

confidence: 99%