654
BIOCHEMICAL SOCIETY TRANSACTIONS
containing the single cysteine residue and each of the two tryptophan residues have been determined and located with respect to the polypeptide-chain termini, by using the sidechain assignment derived from the electron-density map. The three methionine residues are all in the adenine-binding domain, as are the two tryptophan residues. The cysteine residue occurs in the largest CNBr fragment, which has a blocked N-terminal residue and forms almost the whole of the other domain. Both tryptophan residues are located in the C-terminal CNBr fragment, which also appears to contain all the residues involved in catalysis, as is shown in Fig. 1. The precise location of these important residues thus decreases the immediate chemical-sequence problem to one of more manageable proportions. The prospect of the sequence data for the active-centre region, together with the initiation of nuclear-magnetic-resonance studies of the yeast enzyme using all four 31P-labelledsubstrates, has prompted us to re-examine the substrate-binding problem and to extend our structure determination of 0.25 nm (2.5A) resolution. We thank the Science Research Council for financial support. Blake, C. C. F. (1975) Essuys Biochem. 11, 37-79 Blake, C. C. F. & Evans, P. R. (1974) J. Mol. Biol. 84,585-601 Bryant, T. N. (1974) Ph.D. Thesis, University of Bristol Bryant, T. N., Watson, H. C. & Wendell, P. L. (1974) Nature (London) 247, 14-17 Biicher, T. (1947) Biochim. Biophys. Acfa 1,292-314 Cohn, M. (1963) BiochemiJtry 2,623-629 Kulbe, K. D., Nau, H. & Tang, J. J. N. (1976) Abstr. FEBS Meet. 10th
Structure of Cat Muscle Pyruvate Kinase at 0.26nm Resolution DAVID K. STAMMEFLS, MICHAEL LEVINE, DAVID I. STUART and HILARY MUIRHIEAD
Molecular Enzymology Laboratory, Department of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K. Pyruvate kinase (EC 2.7.1.40) catalyses the conversion of phosphoenolpyruvate into pyruvate by the addition of a proton and the loss of a phosphoryl group which is transferred to ADP :
+
+
Phosphoenolpyruvate ADP ,H+
-
L
MEZ+,K+
+
pyruvate ATP
The reaction is essentially irreversible and requires both a bivalent and a univalent cation (Kayne, 1973). Different isoenzymes of pyruvate kinase have been identified. The L-type, present in liver, shows allosteric activation by phosphoenolpyruvate and fructose bisphosphate, anid inhibition by ATP (Tanaka et al., 1967), whereas the M-type, found in mammalian skeletal muscle does not show these regulatory properties. The three-dimensional structure of the M-type enzyme from cat muscle has now been determined at a nominal resolution of 0.26nm. The molecule is a tetramer of approx. mol.wt. 237000 and cont,ains four identical polypeptide chains (Stammers & Muirhead, 1975). The determination of the structure of the enzyme at this resolution means that the conformation of the active site can be investigated and comparisons with other known protein structures can be made. Once the amino acid sequence is known (Harkins & Fothergill, 1977) it should be possible to comment on possible catalytic mechanisms (Mildvan et al., 1976). Cat muscle pyruvate kinase crystallizes as described by Stammers & Muirhead (1975). The crystallographic asymmetric unit is one subunit and the tetramer has crystallographic222symmetry. Rotation-oscillationdatawerecollectedto aresolution o f 0 . 2 6 ~ ~ by using the Arndt-Wonacott camera (Amdt et al., 1973) and phases were determined by using three isomorphous heavy-atom derivatives. These data were combined with preexisting medium-resolution data before calculating the electron-density map. 1977
65 5
568th MEETING, ABERDEEN
Fig. 1. Diagrammatic representation of the three domains of the pyruvate kinase subunit (a) and schematic diagram of the secondary structure of domain A (b) (a) The three intersecting crystallographic dyad axis are shown and the centre of the tetramer is at their point of intersection. X marks the active site and Y the ADP-binding site (Stammers & Muirhead, 1975). (b)The direction of view is looking towards the centre of the molecule from domain B. The cylinders represent a-helices and the arrows strands of 8-sheet. A and B mark connections to and from domain B, C marks the connection to domain C. N marks the N-terminus.
The structure of each subunit consists of three domains; A, B and C (Fig. la). The parts of the linear amino-acid sequence involved in each domain are: I I
I I I I I
f
I
I
4
:
I
\
N-Terminus
I
I I
I
B i I
VOl. 5
A
i 4
C
C-Terminus
656
/A’5h
B’3
/B’2\
A’4
j’l
BIOCHEMICAL SOCIETY TRANSACTIONS
containing the single cysteine residue and each of the two tryptophan residues have been determined and located with respect to the polypeptide-chain termini, by using the sidechain assignment derived from the electron-density map. The three methionine residues are all in the adenine-binding domain, as are the two tryptophan residues. The cysteine residue occurs in the largest CNBr fragment, which has a blocked N-terminal residue and forms almost the whole of the other domain. Both tryptophan residues are located in the C-terminal CNBr fragment, which also appears to contain all the residues involved in catalysis, as is shown in Fig. 1. The precise location of these important residues thus decreases the immediate chemical-sequence problem to one of more manageable proportions. The prospect of the sequence data for the active-centre region, together with the initiation of nuclear-magnetic-resonance studies of the yeast enzyme using all four 31P-labelledsubstrates, has prompted us to re-examine the substrate-binding problem and to extend our structure determination of 0.25 nm (2.5A) resolution. We thank the Science Research Council for financial support. Blake, C. C. F. (1975) Essuys Biochem. 11, 37-79 Blake, C. C. F. & Evans, P. R. (1974) J. Mol. Biol. 84,585-601 Bryant, T. N. (1974) Ph.D. Thesis, University of Bristol Bryant, T. N., Watson, H. C. & Wendell, P. L. (1974) Nature (London) 247, 14-17 Biicher, T. (1947) Biochim. Biophys. Acfa 1,292-314 Cohn, M. (1963) BiochemiJtry 2,623-629 Kulbe, K. D., Nau, H. & Tang, J. J. N. (1976) Abstr. FEBS Meet. 10th
Structure of Cat Muscle Pyruvate Kinase at 0.26nm Resolution DAVID K. STAMMEFLS, MICHAEL LEVINE, DAVID I. STUART and HILARY MUIRHIEAD
Molecular Enzymology Laboratory, Department of Biochemistry, University of Bristol, Bristol BS8 1TD, U.K. Pyruvate kinase (EC 2.7.1.40) catalyses the conversion of phosphoenolpyruvate into pyruvate by the addition of a proton and the loss of a phosphoryl group which is transferred to ADP :
+
+
Phosphoenolpyruvate ADP ,H+
-
L
MEZ+,K+
+
pyruvate ATP
The reaction is essentially irreversible and requires both a bivalent and a univalent cation (Kayne, 1973). Different isoenzymes of pyruvate kinase have been identified. The L-type, present in liver, shows allosteric activation by phosphoenolpyruvate and fructose bisphosphate, anid inhibition by ATP (Tanaka et al., 1967), whereas the M-type, found in mammalian skeletal muscle does not show these regulatory properties. The three-dimensional structure of the M-type enzyme from cat muscle has now been determined at a nominal resolution of 0.26nm. The molecule is a tetramer of approx. mol.wt. 237000 and cont,ains four identical polypeptide chains (Stammers & Muirhead, 1975). The determination of the structure of the enzyme at this resolution means that the conformation of the active site can be investigated and comparisons with other known protein structures can be made. Once the amino acid sequence is known (Harkins & Fothergill, 1977) it should be possible to comment on possible catalytic mechanisms (Mildvan et al., 1976). Cat muscle pyruvate kinase crystallizes as described by Stammers & Muirhead (1975). The crystallographic asymmetric unit is one subunit and the tetramer has crystallographic222symmetry. Rotation-oscillationdatawerecollectedto aresolution o f 0 . 2 6 ~ ~ by using the Arndt-Wonacott camera (Amdt et al., 1973) and phases were determined by using three isomorphous heavy-atom derivatives. These data were combined with preexisting medium-resolution data before calculating the electron-density map. 1977
65 5
568th MEETING, ABERDEEN
Fig. 1. Diagrammatic representation of the three domains of the pyruvate kinase subunit (a) and schematic diagram of the secondary structure of domain A (b) (a) The three intersecting crystallographic dyad axis are shown and the centre of the tetramer is at their point of intersection. X marks the active site and Y the ADP-binding site (Stammers & Muirhead, 1975). (b)The direction of view is looking towards the centre of the molecule from domain B. The cylinders represent a-helices and the arrows strands of 8-sheet. A and B mark connections to and from domain B, C marks the connection to domain C. N marks the N-terminus.
The structure of each subunit consists of three domains; A, B and C (Fig. la). The parts of the linear amino-acid sequence involved in each domain are: I I
I I I I I
f
I
I
4
:
I
\
N-Terminus
I
I I
I
B i I
VOl. 5
A
i 4
C
C-Terminus
656
/A’5h
B’3
/B’2\
A’4
j’l
