Linked Life Data

2684274

Source:http://linkedlifedata.com/resource/pubmed/id/2684274

Statements in which the resource exists as a subject.
PredicateObject
rdf:type
lifeskim:mentions
pubmed:issue
18
pubmed:dateCreated
1990-1-11
pubmed:abstractText
Six individual amino acid substitutions at separate positions in the tertiary structure of subtilisin BPN' (EC 3.4.21.14) were found to increase the stability of this enzyme, as judged by differential scanning calorimetry and decreased rates of thermal inactivation. These stabilizing changes, N218S, G169A, Y217K, M50F, Q206C, and N76D, were discovered through the use of five different investigative approaches: (1) random mutagenesis; (2) design of buried hydrophobic side groups; (3) design of electrostatic interactions at Ca2+ binding sites; (4) sequence homology consensus; and (5) serendipity. Individually, the six amino acid substitutions increase the delta G of unfolding between 0.3 and 1.3 kcal/mol at 58.5 degrees C. The combination of these six individual stabilizing mutations together into one subtilisin BPN' molecule was found to result in approximately independent and additive increases in the delta G of unfolding to give a net increase of 3.8 kcal/mol (58.5 degrees C). Thermodynamic stability was also shown to be related to resistance to irreversible inactivation, which included elevated temperatures (65 degrees C) or extreme alkalinity (pH 12.0). Under these denaturing conditions, the rate of inactivation of the combination variant is approximately 300 times slower than that of the wild-type subtilisin BPN'. A comparison of the 1.8-A-resolution crystal structures of mutant and wild-type enzymes revealed only independent and localized structural changes around the site of the amino acid side group substitutions.(ABSTRACT TRUNCATED AT 250 WORDS)
pubmed:language
eng
pubmed:journal
pubmed:citationSubset
IM
pubmed:chemical
pubmed:status
MEDLINE
pubmed:month
Sep
pubmed:issn
0006-2960
pubmed:author
pubmed:issnType
Print
pubmed:day
5
pubmed:volume
28
pubmed:owner
NLM
pubmed:authorsComplete
Y
pubmed:pagination
7205-13
pubmed:dateRevised
2008-11-21
pubmed:meshHeading
pubmed:year
1989
pubmed:articleTitle
Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding.
pubmed:affiliation
Genex Corporation, Gaithersburg, Maryland 20877.
pubmed:publicationType
Journal Article, Research Support, U.S. Gov't, Non-P.H.S.