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


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 Mn(2+) 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 (10(4)- to 10(6)-fold) reductions in the binding affinity for Mn(2+). Most of the mutations decreased k(cat) for MgPP(i) by 2-3 orders of magnitude when measured with Mn(2+) or Mg(2+) bound to the high-affinity subsite and Mg(2+) bound to both the low-affinity subsite and pyrophosphate. In the E78D variant, the k(cat) for the Mn-bound enzyme was decreased 120-fold, converting bsPPase from an Mn-specific to an Mg-specific enzyme. K(m) values were less affected by the mutations, and, interestingly, were decreased in most cases. Mutations of His(97) and His(98) residues, which lie near the subunit interface, greatly destabilized the bsPPase dimer, whereas most other mutations stabilized it. Mn(2+), in sharp contrast to Mg(2+), 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. Study holds ProTherm entries: 18643, 18644, 18645, 18646, 18647, 18648, 18649, 18650, 18651, 18652, 18653, 18654, 18655, 18656, 18657, 18658, 18659, 18660, 18661, 18662, 18663, 18664, 18665, 18666, 18667, 18668, 18669, 18670, 18671 Extra Details: Enzymes were preincubated with 1.5 mM Mn2+; 13 microM EGTA was added in the experiment Mn(2+) binding affinity; thermostability; active site structure; conservative mutations

Submission Details

ID: NtLjwuTZ3

Submitter: Connie Wang

Submission Date: April 24, 2018, 8:50 p.m.

Version: 1

Publication Details
Halonen P;Tammenkoski M;Niiranen L;Huopalahti S;Parfenyev AN;Goldman A;Baykov A;Lahti R,Biochemistry (2005) Effects of active site mutations on the metal binding affinity, catalytic competence, and stability of the family II pyrophosphatase from Bacillus subtilis. PMID:15751976
Additional Information

Structure view and single mutant data analysis

Study data

No weblogo for data of varying length.
Colors: D E R H K S T N Q A V I L M F Y W C G P
 

Data Distribution

Studies with similar sequences (approximate matches)

Correlation with other assays (exact sequence matches)


Relevant PDB Entries

Structure ID Release Date Resolution Structure Title
1WPN 2004-11-23 1.3 Crystal structure of the N-terminal core of Bacillus subtilis inorganic pyrophosphatase
2HAW 2006-11-14 1.75 Crystal structure of family II Inorganic pyrophosphatase in complex with PNP
1WPM 2004-11-23 2.05 Structure of Bacillus subtilis inorganic pyrophosphatase
2IW4 2006-11-07 2.15 CRYSTAL STRUCTURE OF BASILLUS SUBTILIS FAMILY II INORGANIC PYROPHOSPHATASE MUTANT, H98Q, IN COMPLEX WITH PNP
1K23 2001-10-31 3.0 Inorganic Pyrophosphatase (Family II) from Bacillus subtilis

Relevant UniProtKB Entries

Percent Identity Matching Chains Protein Accession Entry Name
96.4 Manganese-dependent inorganic pyrophosphatase A7ZAR2 PPAC_BACVZ
100.0 Manganese-dependent inorganic pyrophosphatase P37487 PPAC_BACSU