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Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus.
Biochemistry. 1998 Apr 28; 37(17):5939-46.B

Abstract

The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP.

Authors+Show Affiliations

Institute for Protein Research, Osaka University, Suita City, Japan.No affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info availableNo affiliation info available

Pub Type(s)

Journal Article
Research Support, Non-U.S. Gov't

Language

eng

PubMed ID

9558328

Citation

Ogasahara, K, et al. "Electrostatic Stabilization in Methionine Aminopeptidase From Hyperthermophile Pyrococcus Furiosus." Biochemistry, vol. 37, no. 17, 1998, pp. 5939-46.
Ogasahara K, Lapshina EA, Sakai M, et al. Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus. Biochemistry. 1998;37(17):5939-46.
Ogasahara, K., Lapshina, E. A., Sakai, M., Izu, Y., Tsunasawa, S., Kato, I., & Yutani, K. (1998). Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus. Biochemistry, 37(17), 5939-46.
Ogasahara K, et al. Electrostatic Stabilization in Methionine Aminopeptidase From Hyperthermophile Pyrococcus Furiosus. Biochemistry. 1998 Apr 28;37(17):5939-46. PubMed PMID: 9558328.
* Article titles in AMA citation format should be in sentence-case
TY - JOUR T1 - Electrostatic stabilization in methionine aminopeptidase from hyperthermophile Pyrococcus furiosus. AU - Ogasahara,K, AU - Lapshina,E A, AU - Sakai,M, AU - Izu,Y, AU - Tsunasawa,S, AU - Kato,I, AU - Yutani,K, PY - 1998/5/23/pubmed PY - 1998/5/23/medline PY - 1998/5/23/entrez SP - 5939 EP - 46 JF - Biochemistry JO - Biochemistry VL - 37 IS - 17 N2 - The thermostability of methionine aminopeptidase from a hyperthermophile P. furiosus (PfMAP) was extremely high: the denaturation temperature was 106.2 degreesC at pH 10.2. To explore the contribution of electrostatic interaction to the superior thermostability of PfMAP, the thermostability of PfMAP was examined by differential scanning calorimetry (DSC) in various salt concentrations in the acidic region far from the isoelectric point of PfMAP. (1) In 20 mM glycine buffer, the DSC curve of PfMAP exhibited a single peak. Transition temperatures (Tm) were lowered with decreasing pH from 4 to 3. The heat denaturation of PfMAP was not reversible. (2) Denaturation enthalpy (DeltaH) measured at different pHs linearly correlated with Tm up to 102 degreesC, suggesting that the denaturation heat capacity (DeltaCp) for PfMAP is constant up to 100 degreesC. DeltaCp was estimated to be 0.82 J K-1 g-1. (3) In the presence of 10-100 mM KCl at pH 3.2, two peaks appeared on the DSC curves. The first peak shifted to lower temperatures with increasing concentration of KCl and, oppositely, the second one to higher temperatures. It was found that the first and second peaks originated from the heat denaturation of the native form of PfMAP and the melting of the non-native associated form having molten globule-like structure, respectively, judged from the CD spectra and ultracentrifugation analyses. This indicates the following: first, the attractive electrostatic interaction is an important factor in stabilizing the native form of PfMAP; second, the presence of KCl stimulates the formation of the molten globule-like state of PfMAP and stabilizes it. (4) In a comparison of the sequence and crystal structure of PfMAP, which has been recently determined (1xgs.pdb), with those of MAP from Escherichia coli (EcMAP), it was predicted that the extra four short-range ion pairs less than 3 A involved in PfMAP are crucial candidates as determinants for the superior thermostability of PfMAP. SN - 0006-2960 UR - https://www.unboundmedicine.com/medline/citation/9558328/Electrostatic_stabilization_in_methionine_aminopeptidase_from_hyperthermophile_Pyrococcus_furiosus_ L2 - https://doi.org/10.1021/bi973172q DB - PRIME DP - Unbound Medicine ER -