Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis

Pasi Halonen, Marko Tammenkoski, Laila Niiranen, Sauli Huopalahti, Alexey N. Parfenyev, Adrian Goldman, Alexander Baykov (Corresponding Author), Reijo Lahti (Corresponding Author)

Research output: Contribution to journalArticleScientificpeer-review

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Abstract

Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn2+ binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (104- to 106-fold) reductions in the binding affinity for Mn2+. Most of the mutations decreased kcat for MgPPi by 2−3 orders of magnitude when measured with Mn2+ or Mg2+ bound to the high-affinity subsite and Mg2+ bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the kcat for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. Km values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His97 and His98 residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn2+, in sharp contrast to Mg2+, conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.
Original languageEnglish
Pages (from-to)4004 - 4010
Number of pages7
JournalBiochemistry
Volume44
Issue number10
DOIs
Publication statusPublished - 2005
MoE publication typeA1 Journal article-refereed

Fingerprint

Pyrophosphatases
Bacilli
Bacillus subtilis
Mental Competency
Catalytic Domain
Metals
Mutation
Catalysis
Inorganic Pyrophosphatase
Enzymes
Dimers
Bacteria
Viperidae

Keywords

  • pyrophosphatases
  • PPases
  • enzymes
  • catalysts

Cite this

Halonen, P., Tammenkoski, M., Niiranen, L., Huopalahti, S., Parfenyev, A. N., Goldman, A., ... Lahti, R. (2005). Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis. Biochemistry, 44(10), 4004 - 4010. https://doi.org/10.1021/bi047926u
Halonen, Pasi ; Tammenkoski, Marko ; Niiranen, Laila ; Huopalahti, Sauli ; Parfenyev, Alexey N. ; Goldman, Adrian ; Baykov, Alexander ; Lahti, Reijo. / Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis. In: Biochemistry. 2005 ; Vol. 44, No. 10. pp. 4004 - 4010.
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Halonen, P, Tammenkoski, M, Niiranen, L, Huopalahti, S, Parfenyev, AN, Goldman, A, Baykov, A & Lahti, R 2005, 'Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis', Biochemistry, vol. 44, no. 10, pp. 4004 - 4010. https://doi.org/10.1021/bi047926u

Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis. / Halonen, Pasi; Tammenkoski, Marko; Niiranen, Laila; Huopalahti, Sauli; Parfenyev, Alexey N.; Goldman, Adrian; Baykov, Alexander (Corresponding Author); Lahti, Reijo (Corresponding Author).

In: Biochemistry, Vol. 44, No. 10, 2005, p. 4004 - 4010.

Research output: Contribution to journalArticleScientificpeer-review

TY - JOUR

T1 - Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis

AU - Halonen, Pasi

AU - Tammenkoski, Marko

AU - Niiranen, Laila

AU - Huopalahti, Sauli

AU - Parfenyev, Alexey N.

AU - Goldman, Adrian

AU - Baykov, Alexander

AU - Lahti, Reijo

PY - 2005

Y1 - 2005

N2 - Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn2+ binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (104- to 106-fold) reductions in the binding affinity for Mn2+. Most of the mutations decreased kcat for MgPPi by 2−3 orders of magnitude when measured with Mn2+ or Mg2+ bound to the high-affinity subsite and Mg2+ bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the kcat for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. Km values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His97 and His98 residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn2+, in sharp contrast to Mg2+, conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.

AB - Family II inorganic pyrophosphatases (PPases) have been recently found in a variety of bacteria. Their primary and tertiary structures differ from those of the well-known family I PPases, although both have a binuclear metal center directly involved in catalysis. Here, we examined the effects of mutating one Glu, four His, and five Asp residues forming or close to the metal center on Mn2+ binding affinity, catalysis, oligomeric structure, and thermostability of the family II PPase from Bacillus subtilis (bsPPase). Mutations H9Q, D13E, D15E, and D75E in two metal-binding subsites caused profound (104- to 106-fold) reductions in the binding affinity for Mn2+. Most of the mutations decreased kcat for MgPPi by 2−3 orders of magnitude when measured with Mn2+ or Mg2+ bound to the high-affinity subsite and Mg2+ bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the kcat for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. Km values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His97 and His98 residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn2+, in sharp contrast to Mg2+, conferred high thermostability to wild-type bsPPase, although this effect was reduced by all of the mutations except D203E. These results indicate that family II PPases have a more integrated active site structure than family I PPases and are consequently more sensitive to conservative mutations.

KW - pyrophosphatases

KW - PPases

KW - enzymes

KW - catalysts

U2 - 10.1021/bi047926u

DO - 10.1021/bi047926u

M3 - Article

VL - 44

SP - 4004

EP - 4010

JO - Biochemistry

JF - Biochemistry

SN - 0006-2960

IS - 10

ER -