Abstract:
:Soluble inorganic pyrophosphatase (PPase) is one of the better understood phosphoryl-transfer enzymes and is distinctive in having four divalent metal ions at the active site. Here we determine pH profiles for wild-type Saccharomyces cerevisiae PPase (Y-PPase) and for 14 of its active site variants and consider the effects of active site mutation on the pH-independent parameters and acid dissociation constants that characterize these profiles against the framework of the proposed structure of the activated complex. The results obtained (a) support the current mechanistic model in which a hydroxide ion, stabilized by binding to two metal ions at the active site and by an extended system of hydrogen bonds within the active site, is the nucleophile that attacks enzyme-bound inorganic pyrophosphate and (b) provide evidence that the acid group that is necessary for maximal activity is a water molecule coordinated to a third metal ion, as shown by the general rise in the pKa of this group that is a consequence of almost all of the mutations. We further compare the present results to those previously observed for the corresponding mutations in Escherichia coli PPase [E-PPase; Salminen et al. (1995) Biochemistry 34, 782-791]. Such comparison provides a measure of the extent to which different portions of the active site are conserved. We find that some corresponding mutations have different effects on catalytic function, demonstrating that even in the context of very similar active sites, interactions of the mutated site with less well conserved portions of the enzyme, in this case outside the active site, can lead to different outcomes. On the other hand, one region of the active site is highly conserved, suggesting that it may represent a common feature of phosphoryl-transfer enzymes or a vestige of a primitive ur-PPase active site.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Pohjanjoki P,Lahti R,Goldman A,Cooperman BSdoi
10.1021/bi971771rsubject
Has Abstractpub_date
1998-02-17 00:00:00pages
1754-61issue
7eissn
0006-2960issn
1520-4995pii
bi971771rjournal_volume
37pub_type
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