Evolution in glycolysis - Semantic Scholar

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Apr 10, 1987 - (Tolan et ul., 1984); Hum B, human liver (partial sequences) (Costanzo et al., 1983; ... By contrast, the liver (or B) isoenzyme of aldolase is.
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mutagenesis to replace all the wild-type tryptophan residues of the protein, usually by tyrosine. (ii) Measurement of the properties of the tryptophan-less mutant to establish these have not been changed by mutation; (iii) Insertion of a single tryptophan residue a t the site where motion is to be measured. (iv) Characterization of that motion from the changed single-tryptophan fluorescence intensity or anisotropy induced when motion is triggered by substrate-, effector- or inhibitor-binding or by parameters such as temperature, pressure, solvent. viscosity, etc. (v) Repetition of steps (iii) and (iv) with tryptophan probes a t all sites within ii sub-structure until a m a p of the whole domain motion is completed. A similar approach using chemical modification of a uniquely reactive single inserted amino acid (e.g. cysteine) is also possible. Successively replacing the wild-type tryptophan residues (80. 150. 203) of Bucillus slrurothermophilus L D H by tyrosine did not change substrate or coenzyme-binding, regulation by fructose-l,6-bisphosphate,k,;,, or thermal stability (Waldmnn P I ul.. 1987). The gene lacking tryptophan codons was then used (Clarke i’t ul.. 1986) to generate single tryptophan mutants at 106 (Gly + Trp), 216 (Lys + Trp), 237 (Tyr + T r p ) and 248 (Tyr + Trp). These site-directed with the mutants had unchanged enzyme properties exception of 2 16. Removal of a surface ion pair (Lys-216 to Glu-224) reduces the thermal stability from 90 to 70°C and is consistent with this surface loop (210- 224) being a hot spot from which thermal folding and unfolding nucleates. Tryphophan-248 is designed to probe the formation of the Q-axis intersubunit contact, Trp-203 to probe for the P-axis ) Trp-237 as a intersubunit contact (Clarke C I d . , 1 9 8 5 ~ and probe o n the ‘body‘ of the protein for coenzyme loopclosure (Figs. I and 2 ~ ) . The method has been initially tested by mapping the movement of the coenzyme loop (residues 98-1 10) of L D H . The movement by 1.3 nm (defined by the apo- and ternary crystal structures) of this polypeptide loop to close over the surface of coenzyme bound to the active ste of L D H is triggered by the entry of pyruvate into the enzymecoenzyme active site. The movement serves two roles: first, it solvates the bound-nicotinamide coenzyme ring with nonpolar amino acids and is the basis of the oil-water--histidine mechanism of this enzyme (Parker & Holbrook. 1977); more recently Clarke cJt ul. (1986) used site-directed mutagenesis to reveal that loop movement swings Arg-109 into the active centre to polarize the pyruvate carbonyl and to reduce the energy of the transition state. Rates of defined movements have previously been probed: “C-n.m.r. at Cys-165 revealed a rate equal to the reverse V,,, (200s I at room temperature; ~

Waldman et ul., 1986) while the spectrum of nitrotyrosine237 (Parker et ul., 1982, Clarke et ul., 1985h) revealed a faster rate (3000 s I ) at room temperature. We report the use of one cycle of the new method to probe loop-movement (at position 106) t o produce a n initial threepoint low-resolution m a p of the domain movement. Fig. 2u shows a super-imposition of the extreme (apo- and ternary) crystal structures (from M. G. Rossmann’s laboratory via the Brookhaven Protein Data Bank files) into which are built the gene-derived sequence of the bacterial enzyme (J. J. Holbrook, unpublished work). As expected, internalizing T r p a t 106 gives 15% increased fluorescence intensity owing to reduced solvent collision quenching. The firstorder rate of T r p movement is the same as the V,,, both a t 16°C (Fig. 2h; 2.5 s I ) and a t 25°C (250s I ) . The initial three-point m a p of domain movement is shown in Fig. 21, and reveals a t least two components to the motion. More mutant probes will give greater kinetic and structural resolution. The decoupling of these two motions provides an explanation for the stabilization a t 16°C in 30% D M S O of half the enzyme in a superactive form (with k,,, 20 times that of the steady state; Clarke rt ul., 19856) in which Arg-109 has already swung into the active centre. The rate of oxidation of N A D H is then only limited by the fast movement sensed by Tyr-237 after pyruvate has collided with the enzyme (Fig. 2 d ) . Under normal turnover conditions recognition of pyruvate by this enzyme involves at least three steps: bimolecular diffusion into the active site; protein rearrangement around Tyr-237 a t 3000 s I and coenzyme loop closure at 200 s I . ~

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~

Clarke. A. R., Evington. J. R. N.. Dunn. C . R.. Atkinson, T. & Holbrook. J. J. ( 1 9 8 5 ~ Siochim. ) Biophj,.s. Aeru 870. 112 126 Clarke. A. R., Waldman. A. D. B.. Hart. K . & Holbrook. J. J. ( 1985h) Bi(JChit7l. Biophj,.v. A c / u 829. 397 407 Clarke, A. R., Wigley, D. R.. Barstow. D. A,, Atkinson, T., Chia. W. N . & Holbrook, J. J . (1986) No/urc, (I.ondon) 324. 699 702 Fersht. A. R. (19x7) Bioc,/icw. Sot,. T r u m 15. in the press Parker. D. M. & Holbrook. J. J. (1977) in Pyridiw Nuclrwidc Depcvckn/ nc./i~~lroKc’”rr.se.s (Sund. H.. ed.), pp. 485 502. W. de Gruyter. Berlin Parker. D. M.. Jeckel. D. & Holbrook, J. J. (1982) Biochcni. J . 201. 465 471 Waldman. A. D. B.. Birdsall. B.. Roberts. G . C. K . & Holbrook. J. J . ( 1986) Biochini. Bioplij,.~. Acru 870, 102 I I I Waldman. A. D. B..Clarkc. A. R.. Wigley, D. R.. Hart, K . W..Chia, W. N.. Barstow. D. A.. Atkinson. T.. Munro. I . & Holbrook. J. J . (1987) Biodtini. Siop/ijx. ,4i,/u. 913. 66 71 Received 10 April 1987

Evolution in glycolysis LINDA A. F O T H E R G I L L - G I L M O R E I~cyurtnicntof’ Bioihemistrj~,Uniwrsily of’ Edinhurgh, Gcorgci Syuurr. Edinhurgh EH8 Y X D , U . K . Glycolytic enzymes evolve slowly. Comparisons of the primary. tertiary, and quaternary structures of these enzymes are thus particularly informative regarding their evolution and function. In particular, comparisons of tissue-specific and developmental isoenzymes can help to explain their control and organization. More is known about the detailed structures of the 15 enzymes involved in glycolysis than about any other comparable group of enzymes. A remarkable concerted effort has yielded the high resolution crystallographic structures of Abhreviation used: PAM. accepted point mutation

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all of these enzymes. In addition. about 50 complete amino acid sequences are available which in many cases represent sequences of a particular enzyme from a wide range of different organisms. In a few cases, the sequences of tissuespecific isoenzymes have been determined. This wealth of structural information (reviewed by Fothergill-Gilmore. 1986) means that we are in a good position to make the structural comparisons mentioned above. However, it is important to stress that the data are frequently still quite sparse, and some of the generalizations that seem plausible now, may prove to be incorrect as more data became available. Nevertheless, useful points can emerge from the evolutionary comparisons that can be made at the moment. A consideration of the sequences of fructose-1,6bisphosphate aldolase from different organisms and from different mammalian tissues (Table I ) provides a number of

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Table 1. Evolution qf aldolasP The number of residues compared relates only to known, matched sequences; deletions and compositions of unsequenced peptides are not included. The sequences are available in the following publications: Hum A, human muscle (P. S. Freemont, L. Sawyer, B. Dunbar & L. A. Fothergill-Gilmore, unpublished work); Rab A, rabbit muscle (Tolan et ul., 1984); Hum B, human liver (partial sequences) (Costanzo et al., 1983; Besmond r t al., 1983); Rat B, rat liver (Tsutsumi rt al., 1983); D. m d . , D. melanoguster (Malek et ul., 1985); T. hru, T. hrucei (Clayton, 1985). Values for accepted point mutations (PAM) and estimates of times since divergence are from Dayhoff (1978). Entries in parentheses are speculative estimates. Sequences compared

Residues cornpared

Differences

Hum A Rab A Hum B Rat B

363 I64

5 13

D m d . Hum A D.md H u m B D.nir/. Rat B

360 161 360

I07 58 I29

29 36 36

Hum A Hum B Rat B D.mc1

360 162 360 357

I85 83 I92 I R9

51

51 53 53

86 86 92 92

I64 363

59 117

35 32

48 43

PAM

(Oh)

Time since divergence (million years)

P A M ' I 00 million years ~

T.hru. T.hru. T.hru. T.hru.

Hum A Hum B Hum A Rat B

1.4 7.9

clues concerning the evolution of this enzyme, and serves as an example of the type of information that can be gained from sequence comparisons. The observed sequence differences per 100 residues are corrected to PAMs (accepted point mutations) to take account of superimposed and back-mutations. One of the most striking features is that the rate of evolution is very slow. This is particularly true of the mammalian muscle (or A) isoenzyme of aldolase which is tolerating only about 2 PAMs/100 million years. It is thus changing at a much lower rate than, for example, ribonuclease, which has a rate of mutation acceptance about 20 times greater, or trypsin, which is changing about 6 times Faster. By contrast, the liver (or B) isoenzyme of aldolase is changing about 5 times faster than the muscle isoenzyme. The overall rate of evolution of aldolase is about 4-6 PAMs/100 million years, and is typical of most of the glycolytic enzymes. This rate is the same as the small, highly specialized cytochrome molecule. Is i t possible to say anything about the significance of these observations? I t is tempting to speculate that the muscle isoenzyme is so highly conserved because it experiences many constraints in addition to the need to maintain catalytic function. For example, it is present in high concentration in a tissue which is itself evolving only very slowly. Muscle aldolase is known to interact with the highly conserved F-actin molecule (Arnold & Pette, 1968), and with phosphofructokinase (Hofer, 1987). Liver, on the other hand, is a tissue which can change relatively rapidly in response to changes in environment. It is perhaps not surprising that liver isoenzymes evolve more quickly, both because the are 'acquiring' additional properties (as for example, with pyruvate kinase) and because they have fewer constraints. Possibly, this is an indication that glycolytic enzymes in liver interact less with each other and/or with other proteins than d o muscle glycolytic enzymes. Comparison of the sequence of Drosophilu mrlunoguster aldolase with those of the mammalian isoenzymes (Table 1) shows that it is more similar to the muscle isoenzyme. This would indicate that the ancestral form corresponded more closely with the muscle isoenzyme, and that the liver isoenzyme arose by gene duplication. I t seems likely that the rate of aldolase evolution in non-mammalian tissues is 4-5 (3

1.4 8.0 31

50 50

I5 75

I .9

II

900 900 900

4.3 5.6 5.6

( I 500) ( 1500) (1 500)

(5.7) (5.7)

(1500)

(6.1) ( 6 .I )

PAMs/ 100 million years, and that after the gene duplication event, the muscle isoenzyme has evolved more slowly and the liver isoenzyme more rapidly. (This type of information is not apparent from the Trypanosoma hrucei sequences, as the protozoan is too evolutionarily distant, and the isoenzyme differences are swamped by the large number of total differences.) I t appears to be a general feature of muscle isoenzymes that they more closely resemble the enzymes in more primitive organisms than d o liver isoenzymes. This is true both with respect to structure as in the case of aldolase, and with respect to activity as for example with hexokinase and phosphofructokinase. A final important point which comes from the comparisons in Table 1, is that the different isoenzyme sequences from a single species can be strikingly different. Thus the human muscle and human liver aldolase sequences are as divergent from each other as they are from D . melanogustc~r aldolase. This would imply that the gene duplication event giving rise to the different isoenzymes either took place early in vertebrate evolution, or that a more recent gene duplication event was followed by a period of exceptionally rapid change. It should be possible to distinguish between these possibilities by the acquisition of additional sequence information from suitable organisms. The comparisons that have been considered so far have involved the average differences between entire amino acid sequences, and are appropriate for estimating evolutionary divergence. Of course, sequence variations are not randomly distributed, and certain regions can be exceptionally conserved or exceptionally variable. I t is no surprise that amino acid residues required for enzyme activity are highly conserved. I t is perhaps rather less expected to note that the terminal regions can show considerable variability. Fig. 1 presents a graphical comparison of the N-terminal 50 residues of five aldolase sequences. Residues I 20 are particularly variable, and for example, three of the five differences between human and rabbit muscle aldolase involve residues 2-4. Why should the terminal regions be so variable? A possible explanation may relate to the fact that terminal regions are usually not buried. Thus these portions of an enzyme may vary both in amino acid sequences and in length without disrupting the conserved tertiary structure I987

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Class I aldolase sequences

a

z

I

I

I

I

I

10

20

30

40

50

Residue No.

Fig. I . Conrpurison of' the N-trrniinul srquencrs (If uldoluse The sequences of the N-terminal50 residues of five aldolases are compared. The sequences are of aldolasc from human muscle, rabbit muscle, rat liver. D. ndunoguster and T. hrucri. Reference for these sequences are given in Table I .

that is required for activity. In some cases, variation of the terminal regions can be correlated with the 'acquisition' of additional properties. Pyruvate kinase provides a particularly straightforward example. Pyruvate kinase can be isolated from mammalian tissues a s four different isoenzymes which have different properties, reflecting the different metabolic requirements of the tissues. The M I isoenzyme is found in skeletal muscle, and shows predominantly hyperbolic Michaelis-Menten kinetics. The other isoenzymes (M2 in kidney, adipose tissue and lung, L in liver. and R in red blood cells) are all allosterically regulated. and show sigmoidal kinetics with respect to the substrate phosphoenolpyruvate (reviewed by Hall & Cottam, 1978). Liver pyruvate kinase can also be regulated by phosphorylation. which results in a decrease in the affinity for phosphoenolpyruvate, and an increase in the affinity for ATP. The N-terminal region of pyruvate kinase is especially variable (Muirhead i't d., 1986). with the liver isoenzyme being 12 residues longer than the muscle enzyme. The serine residue that is the site of phosphorylation is located in this N-terminal extension, and thus the 'acquistion' o f an additional property can be simply explained. The control of expression of the M 1 and M2 isoenzymes of pyruvate kinase is also of particular interest. and provides a quite diffcrcnt example of how limited sequence variation can correlatc with tissue-specific properties isoenzymes. I t is known that there is only one gene which encodes the M I and M 3 isoenzymes. but two different m R N A s (Noguchi & Tanaka. 1982). The sequences o f t h e two m R N A s are identical except I'or :I cluster of base changes which correspond to I! I amino acid replacements between residues 388--432 (Noguchi ct d . . 1986). These residues comprise a major inter-subunit contact region. and are in a suitable location for mediating the different allosteric properties of the two isoctuymes (Muirhead ct d . , 1986). The M I / M 2 gene apparently has two alternative exons in this region. which are differentially spliced out of the primary R N A transcript in ;I tissue-specific manner. It seems likely that the main way glycolytic enzymes have evolved their tissue-specific properties is by gene duplication and subsequent divergence. Aldolase is typical of most of the glycolytic enzymes in this respect. In addition. pyruvate kinase has evolved by exon duplication and differential processing of the primary transcript. There is yet a third type of evolution in glycolysis, which involves the doubling o f ii gene thereby giving rise to a n enzyme of twice the size. Phosphofructokinase from rabbit muscle is twice the size of the bacterial enzyme, and a comparison of the sequences Vol. 15

shows that the N-terminal and C-terminal halves of the mammalian enzyme are homologous to each other, and to the bacterial enzyme (Poorman r t ul., 1984). The N-terminal half is considerably more like the bacterial enzyme (97 PAMs) than is the C-terminal half (I40 PAMs). and has therefore tolerated fewer mutations. I t is likely that the N-terminal portion has been constrained to retain the catalytic properties of phosphofructokinase, whereas the C-terminal portion has been able to evolve more rapidly, and has thus 'acquired' the additional allosteric properties of the mammalian enzyme. Comparison of the amino acid residues at the ligand binding sites (Hellinga & Evans, 1984) gives further support for this supposition. Thus the catalytic aspartate residue in the fructose-6-phosphate binding site is conserved in phosphofructokinase from two different bacteria and in the N-terminal half of the rabbit enzyme, but is replaced by a serine in the homologous site in the C-terminal half. Similarly, the catalytic arginine in the nucleotide portion of the active site is conserved in the two bacterial phosphofructokinases and in the N-terminal half of the rabbit enzyme, but is replaced by a glycine in the C-terminal half. It is possible that hexokinase has evolved in a similar manner, as the animal enzymes are about twice the size of the yeast enzyme. The determination of the sequence of an animal hexokinase will be required to confirm this suggestion. In conclusion, it can be said that it is possible to gain some insights into the function of glycolytic enzymes in muscle from the evolutionary considerations described above. It is clear, however, that the availability of a few more carefully chosen sequences would be a great help to resolve some of the outstanding questions. Perhaps it will be possible to describe the experimentally elusive interactions between glycolytic eniymes in muscle from a description of particularly conserved regions o n their surfaces. I would like to thank D r J . Ottaway for helpful discussions. and the Wellcome Trust and the Science a n d Engineering Research Council for financial support. Arnold. H. & Pette. I>.(1968) Eur J . H i o d 7 c v i 7 . 6. 163 171 Besmond. C.. Dreyfus. J.-C.. Gregori. C.. Frain. M., Sakin, M . M . . Sala Trepat. J . & Kahn. A . ( 19x3) Bioc~hcw7.Biop/fj'\. Rvs. C'on7t~7iu7. 117. 601 609

Clayton. C. E. (19x5) EAlBO J . 4. 2997 3003 Costnnm. E.., C'astagnoli. L.. Dcnte. L.. Arcari. P.. Smith. M.. Costnnzo. P.. Raugei. G . . Irzo. P.. Pictropaolo. T. C.. Bouguelerct. L.. Cimino. F'.. Salvatore. F. & Cortesc. R . (19x3) E M B O J . 2. 51 61 Dayhofl. M . D. (197X) .4//0\o / ' P r o / p i ~.iS c c / w m c t i n t / S/rrrc./urc. vol. 5. Supplement 3 . National Biomedical Research Foundation. Silver Spring. M D . U.S.A. Fothergill-~;ilmore. L. A . ( 19x6) in .Mii//ic/onuiiti Pro/c+i.s. .Siruc.rirrc, t i n d E,olu/ion (Coggins. J. R . & Hardie. D. G.. eds.). pp. XS 174. Elsevier Biomedical Pres. Amsterdam ilall. E. R . & Cottam, G. L. (1978) I m . J . Rhc'/lc~77.9, 78s 703 Hellinga. H.W . & Evans. P. R. (19x5) Eur. J . Biodwni. 149. 363 373 Ilofcr. [I. W. (19x7) Rioc~/rcwr.Sot,. Trtrm. IS. 982 9x4 Malek. A . A.. Suter. F Z.. Frank. G. & Brcnncr-Holzach, 0. (19x5) BiOCht'/?l.Hi/jp/f I,.?. KC'.\.( ~ O t t 7 f J f / l / f . 126. 1')' 205 Muirhead. H., Clayden. D. A . Barford. D.. Lorimer, C. G.. FothergillGilmore. L. A . Schilt7. E. & Schmitt. W . (19x6) EMBO J . 5 . 475 4x1 Noguchi, T . & Tanaka. T. (19x2) J . Biol. C%c,ni. 257. I I10 I I13 Noguchi. T.. Inoue. H. & Tanaka. T . (19x6) J . B i d . Cl~cw.261. 13x07 13812 Poorman, R . A.. Randolph. A,. Kcnip. R. G. & Heinrikson. R . L. (1984) Na/urc, (London) 309. 467 469 Tolan. D. R., Anisdcn. A . B , Putncy. S. D., Urcdea. M . S . & Penhoet. E. E. ( I 9x4) J . R i d Chcw7. 259, I I27 I I3 I Tsutsumi, K., Mukai. T.. Hidaka. S., Miyagara. H . . Tsutsumi. R., Tanaka. T.. Hori. K . & Ishikawa. K . (19x3) J . Biol. Clicvn. 258. 6537 6542

Received 10 April 19x7