| | |  | | | | Record from Web of Science® | | | | | | |
| INTERACTION OF BARNASE WITH ITS POLYPEPTIDE INHIBITOR BARSTAR STUDIED BY PROTEIN ENGINEERING |
|
|
| Author(s): SCHREIBER G, FERSHT AR |
| Source: BIOCHEMISTRY Volume: 32 Issue: 19 Pages: 5145-5150 Published: MAY 18 1993 |
| Times Cited: 157 References: 19 |
| Abstract: Barnase, an extracellular ribonuclease of Bacillus amyloliquefaciens, forms a very tight complex with its intracellular polypeptide inhibitor barstar. At pH 8, the values for the rate constants k1 (association) and k-1 (dissociation) are 6.0 x 10(8) s-1 M-1 and 8.0 X 10-6 s 1, respectively. The value of K(i), the dissociation constant of barstar and barnase, calculated from the ratio k-1/k1 is 1.3 X 10(-14) M, which corresponds to a DELTAG of -18.9 kcal/mol at 25-degrees-C. The dissociation constant increases with decreasing pH according to the ionization of an acid in free barnase of pK(a) 6.4, with very weak, if any, binding to the protonated form. This pH dependence for dissociation of the complex can be attributed almost entirely to residue His102 in barnase, as determined by a His102-->Ala mutation. Analysis of the pH dependence of the kinetic constants indicates that binding is, at least, a two-step process. The first, and rate-determining, step is association at close to the diffusion-controlled rate. There is then the precise docking of the complex. The value of K(i) increases to 2.4 X 10(-11) M in the presence of 500 mM NaCl, and to 1.6 x 10(-11) M at pH 5 (100 mM NaCl). The binding site of barstar on barnase was mapped by measuring the values of K(i) for a broad range of site-specific mutants of barnase. Mutagenesis of residues Lys27, Arg59, Arg87, and His102 to Ala increases the values of K(i) by a factor of 104. These same residues were found previously to be important in the catalytic activity of barnase. |
| Document Type: Article |
| Language: English |
Addresses:
1. MRC, CAMBRIDGE CTR PROT ENGN, MRC, PROT FUNCT & DESIGN UNIT, HILLS RD, CAMBRIDGE CB2 2QH, ENGLAND |
| Publisher: AMER CHEMICAL SOC, 1155 16TH ST, NW, WASHINGTON, DC 20036 |
| Subject Category: Biochemistry & Molecular Biology |
| IDS Number: LC721 |
| ISSN: 0006-2960 |
|
| | | | | | | | | Record from Web of Science® | | | | | | | | | |