Large increases in general stability for subtilisin BPN' through incremental changes in the free energy of unfolding

Abstract

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)

Factors effecting the thermostability of cysteine proteinases from Carica papaya

“…An explanation for a cysteine-mediated increase in chymopapain stability could be similar to that discussed by Pantoliano et al (1989) in a study on modification of stability by disulphide-bond engineering using subtilisin BPN' . In their study, the mutation Gln206Cys, forming an unpaired cysteine, conferred an increase in stability of 5.2 KJ .…”

“…In their study, the mutation Gln206Cys, forming an unpaired cysteine, conferred an increase in stability of 5.2 KJ . mol-' over the wild type with an increase in t,,, of 4.7"C. In their biochemical and crystallographic examination of the mutations, Pantoliano et al (1989) determined that, during purification, the extra cysteine of the Gln206Cys mutant was isolated as a cysteine persulphide Cys-S-SH or C-S-S-. This persulphide form stabilised the molecule due to favourable --) papaya proteinase 3.…”

“…Authors have used this information to engineer stability mutations based on hydrophobic packing and helix stabilisation (Imanaka et al, 1986), electrostatic interactions and helix stabilisation (Pickersgill et al, 1991b ;Goodenough et al, 1991) hydrophobic interactions (Kellis et al, 1988;Shortle et al, 1990), disulphide-bond engineering (Pantoliano et al, 1989), solvent inaccessibility (Green et al, 1992) and folding entropies (Matthews et al, 1987).…”

The denaturation and degradation of stable enzymes at high temperatures

“…On the other hand, quite extensive efforts to use single point mutations to enhance protein stability have been relatively unsuccessful in increasing half-lives by amounts corresponding to stabilizing energy differences (see above and [20]) of as much as 5 kJ:mol −" . Nevertheless, accumulated point mutations have led to stability increases of more than 15 kJ:mol −" [42], and there does not seem to be any theoretical objection to proteins of very much greater conformational stability than those currently known.…”

Context‐dependent protein stabilization by methionine‐to‐leucine substitution shown in T4 lysozyme

“…The key question is whether leucine side chains can be readily accommodated at methionine sites or whether there will be steric interference that will offset the expected benefit of the enhanced transfer free energy and reduced entropic cost outlined above. It was previously shown, for example, that a methionine-to-phenylalanine substitution stabilized subtilisin BPN (Pantoliano et al, 1989) but the Met-to-Leu replacement at site 102 in T4 lysozyme was somewhat destabilizing (Hurley et al, 1992). A Met-to-Leu replacement in Stuphylococcul nucleuse was predicted to increase stability by 1.6 kcal/mol (Yamaotsu et al, 1993) but actually decreased stability by 0.8 kcal/mol (Spencer & Stites, 1996).…”