Download the phosphoglycerate mutase family studied by protein engineering

257
632nd MEETING, CORK
Mutase versus synthase: the phosphoglycerate mutase family studied by protein engineering
MALCOLM F. WHITE and
LINDA A. FOTHERGILL-GILMORE
Ilepurtrnetit of Biochemistry, Utiiversity of Edirihiirgh, George
Syiiure, Edirihirrgh EH8 9XD. U.K.
The enzyme phosphoglycerate mutase ( E C 5.4.2.1) catalyses
the interconversion of 2- and 3-phosphoglycerate in the
glycolytic/gluconeogenic pathways. This enzyme has been
very well characterized, particularly the enzyme from
Succhuromyces cerevisiue whose amino acid sequence and
high-resolution crystal structure have been determined [ I . 21.
A detailed catalytic mechanism has been postulated based o n
this structural and also kinetic information (see [3] for a
recent review). Briefly, a round of catalysis is initiated when
an active-site histidine (His-8) is phosphorylated by the
cofactor 2.3-bisphosphoglycerate. A substrate molecule can
then bind. and phospho-transfer takes place via a ping-pong
mechanism. The flexible C-terminal tail is thought to exclude
water and help anchor intermediates at the active site. After
release of product the enzyme remains phosphorylated and
catalytically competent. The occasional hydrolysis of the
phospho-enzyme accounts for the low 2,3-bisphosphoglycerate phosphatase activity associated with the enzyme.
The wealth of information available for this enzyme,
together with its relatively simple reaction mechanism,
means that it is an excellent candidate for further study using
the technique of site-directed mutagenesis coupled with
extensive kinetic and structural characterization of mutants.
There are two main areas where site-directed mutagcnesis
could yield useful information:
( a ) The enzyme bisphosphoglycerate mutase ( E C 5.4.2.4/
EC 3.1.3.13), which catalyses the synthesis of 2,3-bisphosphoglycerate from 1,3-bisphosphogIycerate (‘synthase’
activity) in erythrocytes, is closely related to phosphoglycerate mutase, sharing approx. 50% sequence identity.
Indeed the two enzymes catalyse the same three reactions,
although at vastly different relative rates. The residues at the
active site are highly conserved, with certain exceptions.
Most notably serine-1 1, a probable phospho-ligand in
phosphoglycerate mutase, is a glycine in bisphosphoglycerate
mutase. Could this replacement account for the differences
in catalytic rates observed for the two enzymes?
(b)The C-terminal tail does not adopt a regular conformation in crystals (presumably due to its mobility), but modelbuilding [2] has shown that the C-terminal lysine could
provide another phospho-ligand at the active site during
catalysis. The tail is known to be essential for activity; what
effect would the loss of the C-terminal lysine have on the
enzyme activity?
The gene encoding yeast phosphoglycerate mutase has
recently been identified and sequenced [ 11. The chromosomal copy of the phosphoglycerate mutase gene has been
deleted from the yeast strain DBY747 to produce the strain
DBYgpm-, which is unable to utilize glucose as a carbon/
energy source, but can grow in media containing ethanol and
glycerol. Two mutant forms of the enzyme, one with a serineAbbreviations used: S1 lG, serine-1 1 to glycine; K246G, lysine246 to glycine.
VOI. 18
Table I . Kinetic purirtri(~ter.s
!Or wild-type nnd mrturii phosphoglycwute inutuses
Mutase activity was measured in the direction 3-phosphoglycerate to 2-phosphoglycerate using an assay coupled through
enolase, pyruvate kinase and lactate dehydrogenase to the oxidation of NADH. Synthase activity was measured by the
method given in 141, and phosphatase activity (unstimulated by
2-phosphoglycollatei by the method given in 151. Phosphate
determination was according t o 161.
Wild type
4,,
(PMI
3-Phosphogl ycerate
2-Phosphogl ycerate
7.3-Bisphosphoglyccr~~~~
k,,, ( \ ‘ 1
Mutaw
Synthase
Phosphataw
650
41
.s
3x3
0.022
0.007
K246G
SI lC
680
670
67
48
60
3
324
0.032
0.007
7. I
0.020
0.0005
1 1 to glycine (S1 l G ) substitution and the other with a lysine246 to glycine (K246G) substitution, have been constructed
and expressed in the yeast strain DBYgpm-. The kinetic
parameters of the wild-type and mutant forms of the enzyme
are compared in Table 1.
The K246G replacement does not appear to have significantly altered any of the kinetic parameters of the enzyme,
and thus the C-terminal lysine does not appear to play a
major role in the catalytic mechanism. In the S11G mutant
the mutase and phosphatase activities, which require 2,3-bisphosphoglycerate as a phospho-donor, are decreased by
99.5 and 94%, respectively. These changes can be ascribed
to a tenfold decrease in the enzyme’s affinity for 2,3-bisphosphoglycerate, presumably due to the loss of the serine11 phospho-ligand. In contrast, the synthase activity, which
requires 1,3-bisphosphogIycerate as a phospho-donor, is
unchanged in the S11G mutant. These observations shed
light on the molecular basis for the ‘mutase versus synthase’
relationship in the phosphoglycerate mutase family.
We thank the Science and Engineering Research Council and the
Wellcome Trust for financial support.
1. White, M. F. & Fothergill-Gilmore, L. A. ( 1988) FE5.S Left. 229,
383-387
2. Winn, S. I., Watson, H. C., Harkins, R. N. & Fothergill, L. A.
( 1981 ) Philos. Tram. R. Soc. London B 293, 12 1 - 130
3. Fothergill-Gilrnore, L. A. & Watson, H. C. (1989)Adv. Enzymol.
62,227-313
4. Laforet, M. T., Butterfield, J. B. & Alpers, J. B. ( I 074) Arch.
Biochern. Biophy~.165, 1 79- 187
5. Sasaki, R., Hirose, M., Sugirnoto, E. & Chiba, H. ( 197 1 ) Biochim.
Biophys. Aria 221, 584-594
6. Lebel, D., Pokier, G. G. & Beaudoin, A. R. (1978) Anal.
Biochem. 85,86-89
Received 19 September 1989