Composite interference optics for the analytical ultracentrifuge

Soluble inorganic pyrophosphatases (PPases) comprise two evolutionarily unrelated families (I and II). These two families have different specificities for metal cofactors, which is thought to be because of the fact that family II PPases have three active site histidines, whereas family I PPases have none. Here, we report the structural and functional characterization of a unique family I PPase from Mycobacterium tuberculosis (mtPPase) that has two His residues (His 21 and His 86 ) in the active site. The 1.3-Å three-dimensional structure of mtPPase shows that His 86 directly interacts with bound sulfate, which mimics the product phosphate. Otherwise, mtPPase is structurally very similar to the well studied family I hexameric PPase from Escherichia coli, although mtPPase lacks the intersubunit metal binding site found in E. coli PPase. The cofactor specificity of mtPPase resembles that of E. coli PPase in that it has high activity in the presence of Mg 2؉ , but it differs from the E. coli enzyme and family II PPases because it has much lower activity in the presence of Mn 2؉ or Zn 2؉ . Replacements of His 21 and His 86 in mtPPase with the residues found in the corresponding positions of E. coli PPase had either no effect on the Mg 2؉ -and Mn 2؉ -supported reactions (H86K) or reduced Mg 2؉ -supported activity (H21K). However, both replacements markedly increased the Zn 2؉ -supported activity of mtPPase (up to 11-fold). In the double mutant, Zn 2؉ was a 2.5-fold better cofactor than Mg 2؉ . These results show that the His residues in mtPPase are not essential for catalysis, although they determine cofactor specificity. Soluble inorganic pyrophosphatase (PPase)3 (EC 3.6.1.1), is an essential metal-dependent enzyme that converts pyrophosphate into orthophosphate. This simple reaction provides a thermodynamic pull for many biosynthetic reactions that yield pyrophosphate as a byproduct (1). Soluble PPases belong to two nonhomologous families (2, 3): family I PPases, which are fairly widespread in all types of organisms, and family II PPases, which are exclusive to bacteria. In eubacteria and archaebacteria, the family I PPases are usually homohexamers, whereas in eukaryotes, they are homodimers. In contrast, all family II PPases are homodimers. The subunit size is generally 19 -22 kDa in hexameric PPases and 31-34 kDa in dimeric forms. Despite the variability of the subunit size in family I PPases, their active site cavities are formed by the same 13 functionally important polar residues, which is reflected in the conservation of the catalytic mechanism (4, 5). Although no sequence or overall structural similarity is observed between the two families, there is a striking similarity in the spatial arrangement of six key active site residues, a remarkable example of convergent enzyme evolution (6, 7).Functionally, family II differs from family I in its preference for Mn 2ϩ over Mg 2ϩ as a cofactor. Mg 2ϩ is a better cofactor for family I PPases, and it also activates family II PPases to the same extent. However, Mn 2ϩ co...

Section: Methodsmentioning

confidence: 99%

An Unusual, His-dependent Family I Pyrophosphatase from Mycobacterium tuberculosis

Tammenkoski

Benini

Magretova

et al. 2005

“…Protein Purification-Sg-PPase and Sm-PPase were purified to homogeneity by DEAE fast flow ion exchange column chromatography in 20 SO 4 in the buffer used for the ion exchange chromatography and eluted with a downward gradient of (NH 4 ) 2 SO 4 concentration. Enzyme purity was checked by electrophoresis in 8 -25% gradient polyacrylamide gels in the presence of 0.55% SDS using the Phast System (Amersham Pharmacia Biotech).…”

Section: Methodsmentioning

confidence: 99%

“…Sedimentation velocity was measured at 48,000 -60,000 rpm, and the sedimentation coefficient, s 20,w , was calculated using a standard procedure (20). Sedimentation equilibrium was attained at 16,000 rpm for 16 h or at 24,000 rpm for 10 h, and the molecular mass was calculated according to Chernyak and Magretova (21).…”

Section: Methodsmentioning

confidence: 99%

Quaternary Structure and Metal Ion Requirement of Family II Pyrophosphatases from Bacillus subtilis,Streptococcus gordonii, and Streptococcus mutans

Parfenyev

Salminen

Halonen

et al. 2001

137

Pyrophosphatase (PPase) from Bacillus subtilis has recently been found to be the first example of a family II soluble PPase with a unique requirement for Mn 2؉ . In the present work, we cloned and overexpressed in Escherichia coli putative genes for two more family II PPases (from Streptococcus mutans and Streptococcus gordonii), isolated the recombinant proteins, and showed them to be highly specific and active PPases (catalytic constants of 1700 -3300 s ؊1 at 25°C in comparison with 200 -400 s ؊1 for family I). All three family II PPases were found to be dimeric manganese metalloenzymes, dissociating into much less active monomers upon removal of Mn 2؉ . The dimers were found to have one high affinity manganese-specific site (K d of 0.2-3 nM for Mn 2؉ and 10 -80 M for Mg 2؉ ) and two or three moderate affinity sites (K d ϳ 1 mM for both cations) per subunit. Mn 2؉ binding to the high affinity site, which occurs with a half-time of less than 10 s at 1.5 mM Mn 2؉ , dramatically shifts the monomer 7 dimer equilibrium in the direction of the dimer, further activates the dimer, and allows substantial activity (60 -180 s ؊1 ) against calcium pyrophosphate, a potent inhibitor of family I PPases.

“…Analytical ultracentrifugation was carried out in a Spinco E instrument (BeckmanSpinco), with scanning at 280 nm. Sedimentation velocity was measured at 48,000 rpm, and the sedimentation coefficient, s 20,w , was calculated using standard procedures (21). A partial specific volume of 0.730 cm 3 /g at 25°C was calculated from the amino acid composition.…”

Section: Methodsmentioning

confidence: 99%

Modulation of Dimer Stability in Yeast Pyrophosphatase by Mutations at the Subunit Interface and Ligand Binding to the Active Site

Salminen

Parfenyev

Salli

et al. 2002

Yeast (Saccharomyces cerevisiae) pyrophosphatase (YPPase) is a tight homodimer with two active sites separated in space from the subunit interface. The present study addresses the effects of mutation of four amino acid residues at the subunit interface on dimer stability and catalytic activity. The W52S variant of Y-PPase is monomeric up to an enzyme concentration of 300 M, whereas R51S, H87T, and W279S variants produce monomer only in dilute solutions at pH > 8.5, as revealed by sedimentation, gel electrophoresis, and activity measurements. Monomeric Y-PPase is considerably more sensitive to the SH reagents N-ethylmaleimide and phydroxymercurobenzosulfonate than the dimeric protein. Additionally, replacement of a single cysteine residue (Cys 83 ), which is not part of the subunit interface or active site, with Ser resulted in insensitivity of the monomer to SH reagents and stabilization against spontaneous inactivation during storage. Active site ligands (Mg 2؉ cofactor, P i product, and the PP i analog imidodiphosphate) stabilized the W279S dimer versus monomer predominantly by decreasing the rate of dimer to monomer conversion. The monomeric protein exhibited a markedly increased (5-9-fold) Michaelis constant, whereas k cat remained virtually unchanged, compared with dimer. These results indicate that dimerization of Y-PPase improves its substrate binding performance and, conversely, that active site adjustment through cofactor, product, or substrate binding strengthens intersubunit interactions. Both effects appear to be mediated by a conformational change involving the C-terminal segment that generally shields the Cys 83 residue in the dimer.