Theoretical calculation of pKa reveals an important role of Arg205 in the activity and stability of Streptomyces sp. N174 chitosanase.
Based on the crystal structure of chitosanase from Streptomyces sp. N174, we have calculated theoretical pK(a) values of the ionizable groups of this protein using a combination of the boundary element method and continuum electrostatics. The pK(a) value obtained for Arg(205), which is located in the catalytic cleft, was abnormally high (>20.0), indicating that the guanidyl group may interact strongly with nearby charges. Chitosanases possessing mutations in this position (R205A, R205H, and R205Y), produced by Streptomyces lividans expression system, were found to have less than 0.3% of the activity of the wild type enzyme and to possess thermal stabilities 4-5 kcal/mol lower than that of the wild type protein. In the crystal structure, the Arg(205) side chain is in close proximity to the Asp(145) side chain (theoretical pK(a), -1.6), which is in turn close to the Arg(190) side chain (theoretical pK(a), 17.7). These theoretical pK(a) values are abnormal, suggesting that both of these residues may participate in the Arg(205) interaction network. Activity and stability experiments using Asp(145)- and Arg(190)-mutated chitosanases (D145A and R190A) provide experimental data supporting the hypothesis derived from the theoretical pK(a) data and prompt the conclusion that Arg(205) forms a strong interaction network with Asp(145) and Arg(190) that stabilizes the catalytic cleft.
Fukamizo
T
T
Laboratory of Biophysical Chemistry, Faculty of Agriculture, Kinki University, Nara, Japan. fukamizo@nara.kindai.ac.jp
Juffer
A H
AH
Vogel
H J
HJ
Honda
Y
Y
Tremblay
H
H
Boucher
I
I
Neugebauer
W A
WA
Brzezinski
R
R
D000595:Amino Acid Sequence; D000596:Amino Acids / Q000737:chemistry; D001120:Arginine / Q000502:physiology*; D020134:Catalytic Domain; D002942:Circular Dichroism; D018360:Crystallography, X-Ray; D004926:Escherichia coli / Q000378:metabolism; D005944:Glucosamine / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000737:chemistry* / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008958:Models, Molecular; D008969:Molecular Sequence Data; D016297:Mutagenesis, Site-Directed; D010957:Plasmids / Q000378:metabolism; D017510:Protein Folding; D017386:Sequence Homology, Amino Acid; D013302:Streptomyces / Q000201:enzymology*; D013696:Temperature; D013816:Thermodynamics; D013997:Time Factors
D000596:Amino Acids; D001120:Arginine; D006026:Glycoside Hydrolases; C019868:chitosanase; D005944:Glucosamine