FUNCIONARIOS, SERVIDORES Y USUARIOS
4.2. Discusión de Resultados
Most of the mechanistic information on these proteins stem from mutagenesis studies of bacteriophage X phosphatase and purple acid phosphatase, phosphatases with substantial homology to the catalytic domains of PPl, PP2A and PP2B^'* □ Catalytic site - solution of the X-ray crystal structures of PPU /'"’^ the PP2B holoenzyme^^ and the PP2B complex with FK506/FKBP12,^^‘"^ provide an understanding of some of the fundamental properties of these proteins, namely molecular mechanisms of catalysis, substrate recognition and regulation.
The first co-crystallisation of the catalytic subunit of PPl complexed to microcystin 2 was reported in 1994 by Barford et cil^^ The crystals were reported to be orthorhombic at a resolution of 2.8 Â. Later, the crystal structure of the mammalian protein phosphatase-1, PPL, complexed with the hepatoto.xin, microcystin, at 2.1Â resolution, was delineated by Goldberg et In the same year, the catalytic subunit of human PPLy and its complex with tungstate at 2.5
a
was reported by Egloff et al.^^Recently, Egloff et reported the crystal structure of PPL with the M-subunit at 3.0
a.
The structure of PP2B was determined by Griffith et al. at 2.5
a
as a complex containing both, FK506 3 and the FK506 binding protein (FKBP).'^ The same complex was also described by Kissinger et al. who also reported the crystal structure of human PP2B heterodimer at 2.1 A.^^The catalytic domains of PPl and PP2B adopt an identical core structure based on a central distorted (3-sandwich of 11 (3-strands surrounded on one side by seven a-helices and a three-stranded g-sheet (see Fig. 1.6).^^
In t r o d u c t i o n P/Uîi' 22
H173 M„
, 'V ■ H24S D M
Figure 1.6: Piew o f the catalytic suhunit o f PPl y onto the catalytic-site channel
Metal ions are shown as green spheres within a hi nuclear metal centre to which a tungstate ion (an analogue o f phosphate) is co-ordinated. Regions o f the polypeptide which correspond to the tht ee invariant secf uence motifs present in
the PPP family o f phosphatases and other metallophosphoesterases, such as X bacteriophage phosphatase and kidney bean purple acid phosphatase, are shown
in red. The C a positions o f residues which co-ordinate the divcdent metal ions and tungstate ion are indicated as blue and red spheres.
I n t r o d u c t i o n p a g e 23
The interface of the two (3-sheets at the top of the P-sandwich creates a shallow channel. Three parallel P-strands of sheet I constitute a mononucleotide-binding domain with the secondary structure organisation P-a-P-a-p. The three invariant sequence motifs form the loops connecting with the carboxy terminus of the p-strand with a-helices (see Fig. 1.7). These loops, together with those emanating from the carboxy terminus of two p-strands of the opposite p-sheet, provide the catalytic residues. This scaffolding contains the Ser/Thr protein phosphatases signature motif identified during studies with bacteriophage X phosphatase.^^ Ser/Thr protein phosphatases are metalloenzymes^^‘^^’^“ and two divalent metal ions (Mn^" and Fe^" in PPl, Zn"^ and Fe^" in PP2B) at the centre of the catalytic site are co-ordinated by Asp64, Asp92, Asnl24 and His66 of the mononucleotide-binding motif and His 173 and His248 of sheet 2 (Note: Residue numbers refer to PPl].
INTRODUCTION P/UîK 24 Asn 124 ;i
^
\ v
A ^
-X L V r ^ - - - - (...r " ...! " " V ’\ I ■ Ar-% »\ (b) Asp95 His 125 Asn124 O * 1418248 / N His 173 Tyr272 Asp64 Hls66Figure 1.7 (a) Detailed view o f the catalytic site o f PPl in the presence o f a tungstate ion, an analogue o f phosphate. One o f the two water molecules shown co-ordinated to the metal ions is proposed to act
as a nucleophile during the catalytic reaction
(b) Schematic o f the reaction mechanism catalysed by PPPs.
The side chain of Asp92 and a water molecule bridge both metal ions to form a binuclear metal centre. A second metal-bound water molecule is co-ordinated to the Fe^ ion. The catalytic sites of two of the phosphatase ciystal structures contain bound oxyanions; tungstate in PPlyl and phosphate in one of the PP2B structures70
□ Mechanism - a catalytic mechanism, suggested by the X-ray structures, is entirely consistent with site-directed mutagensis performed on PPl’^ and the X
bacteriophage phosphatase.^** A metal-bound water molecule acts as a nucleophile to attack the phosphorous atom of a phosphate group in an Sn2 mechanism.
I n t r o d u c t i o n p a g e 25
Metals assist in this catalysis in two ways;
® as Lewis acids to enhance the nucleophilicity of the metal-bound water; and ® by enhancing the electrophilicity of the phosphorous atom.
The sidechain of His 125 probably donates a proton to the leaving-group oxygen of a Ser or Thr sidechain. Such a role for His 125 is consistent with the loss of catalytic activity of PPl His 125 mutants^’ and mutations of the equivalent residue of X phosphatase.^** Other mutational studies have shown replacement of any of the metal-co-ordinating residues in PPl and X PPase leads to either severe loss in activity or production of insoluble proteins during over-production in E. coli. The proposal for a single-step déphosphorylation reaction is consistent with earlier data suggesting PP2B cannot catalyse //wzsphosphorylation reactions^^ and the fact phosphorylenzyme intermediates do not appear to form during the reaction catalysed by PPPs.