the peak eluted with ethylene glycol does not react and the supernatant is uncoloured (Fig 2). When the same samples are allowed to diffuse into the agar plates containing starch, the first peak gives a clear halo indicating the presence of a- or a- and 13- amylase.

a b c d e f

Figure 2 Differentiation of a-amylase and f~-amylase activities by Phadebas amylase test. (a) blank with water; (b) malt extract; (c, d, e) different dilutions of peak I (Fig 1); (f) peak II (Fig 1)

The second peak gives a small pink halo typical from 13- amylase (Fig 3). Thus, the peak eluted by ethylene glycol contains 13-amylase and the peak eluted by salt deletion mainly contains a-amylase.

Figure 3 Differentiation of a-amylase and 13-amylase activities using the agar diffusion method. Left: malt extract; right: peak I; top: sweet potato 13-amylase standard; bottom: peak II.

It is important to point out that Phenyl-Sepharose beds may be re-used many times after being thoroughly washed on a sintered glass filter with distilled water, followed by ethanol and n- butanol. After a few minutes, the gel can be treated with these in the reverse sequence and stored in the adsorption buffer.

Conclusions The resolution of malt extract in a- and 13-amylasic fractions is of particular value as a teaching laboratory exercise because it illustrates several biochemical concepts. It shows, as expected, that the hydrophobic interactions are stronger at high ionic strength, so the adsorption to a hydrophobic adsorbent may be conveniently performed after a salt precipitation or an ion- exchange chromatography step.

The conditions used for the elution show how different parameters (hydrophobicity, polarity of the environment) infuence the interaction between an amphiphilic bed and the adsorbed proteins.

The techniques used to distinguish between a- and 13-amylase activities give the students an idea of two ways by which the

45

differentiation can be carried out. Small regenerable gel beds are used allowing the saving of materials and money.

Acknowledgements We thank IPICS, Uppsala University, Sweden and PEDECIBA (Programa para el Desarrollo de las Ciencias B~isicas), Uruguay for financial support. We are indebted to Ulf Sthfil for fruitful comments and Dr Jeffrey Martin for linguistic revision

References J Daussant, J (1987) in 'Advances in Cereal Science and Technology', vol 9, edited by Pomeranz, Y, 47-60

2Friedberg, F (1985) Biochem Educ 13, 105-107 3Bernfeld, P (1955) in Methods in Enzymology, vol 1, edited by Colowick, S P and Kaplan, N O, 149-150

aphadebas Amylase Test, Pharmacia Diagnostics (1983) 5Briggs, B A (1962) J Inst Brew 68, 27-32 6Hjert6n, S (1980) in 'Methods of Biochemical Analysis', 27, 89-108 7Eriksson, K O (1989) in 'Protein Purification', edited by Janson, J C and Ryd6n, L, VCH, New York, pp 207-226

Molecular Evolution: The Origin of Glycolysis

SIMON POTTER and LINDA A FOTHERGILL- GILMORE

Department o f Biochemistry University of Edinburgh George Square Edinburgh EH8 9XD, Scotland

Introduction Over the past decade or so, a great deal of information about how individual enzymes evolve has been gathered, largely from comparisons of protein and gene structures. These comparisons are facilitated using sequence databases which, to date, hold upwards of thirty million nucleotides of information. It is now understood, for example, that enzyme evolution occurs on various levels ranging from single amino acid changes through insertions, deletions and exon-shufflin~ to gene duplication/ fusion and lateral gene transfer events. ' Unfortunately, much less is known about the evolution of the metabolic pathways of which these enzymes are often a part.

A good case in point is the glycolytic pathway. This sequence of metabolic reactions is catalysed by ten enzymes and is unique in terms of the amount of information available about it. Sequences of all ten enzymes are available from a variety of sources as are their crystal structures. 2 This information has revealed some interesting interrelationships between the enzymes which may begin to shed some light on how the glycolytic pathway as a whole may have evolved. Some of the most interesting of these insights have arisen as a result of studies on the glycolytic enzymes of the archaea, a group of organisms originally distinguished by their ability to exist in extreme habitats: methanogens, which live in badly aerated swampy areas and the ruminant gut; halophiles, which live in areas of high salt content or low water activity, and thermophiles, which live in areas of high temperature such as hot springs. They are all prokaryotic in nature and organisation but possess macro- molecular constituents which more closely resemble their eukaryotic counterpar ts ) In fact, they are now recognised as constituting a third taxonomic superkingdom or 'domain' of equal ranking with the bacteria and the eukarya (note: throu~h- out this article, the new taxonomic terminology of Woese et at" is used).

BIOCHEMICAL EDUCATION 21(1) 1993

46

Use of computers Before discussing the information gleaned from comparative studies of archaeal glycolytic enzymes and their bacterial and eukaryal counterparts, it will be helpful to review briefly the theoretical background to the studies. If two proteins are homologous (ie are derived from a common ancestor), when their sequences are aligned they should be over 25% identical. The sequences may be aligned using computer programs such as CLUSTAL 5 which aim to produce maximum identity between sequences by introducing small gaps into them. For an example of the use of gapping in sequence alignment see Fig 1 which shows two alignments of the a and 13 chains of haemoglobin, one with gaps and the other without. 6 The threshold value of 25% identity is an average of the values that would be expected were two unrelated protein sequences to be aligned with gaps. The reason for this is that alignment programs allow gaps to be incorporated into either sequence if, by doing so, the alignment is significantly improved. An appropriate gap penalty is imposed in order that gapping not be unrestrained. For example, gaps are seldom introduced into regions of probable secondary structure such as a-helices. If gapping were not allowed, two random sequences would be about 5-6% identical. If two sequences show identity above the threshold value they are said to be homologous. If they show identity around the threshold value or below it, it is probable that they are not homologous (although it is impossible to say that with certainty as they may be distantly related to one another, retaining only a few vital structural and functional regions).

In the case of multiple sequence alignments, which can be performed by programs such as CLUSTAL, it is possible to construct a matrix of percentage sequence identities (or differ- ences) which relates each protein to its counterparts from other organisms. Although more and more proteins are being sub- jected to this type of analysis, the pioneering work of Carl Woese and his co-workers was performed using alignments of RNA sequences rather than proteins. A matrix of rRNA differences may be used to generate a phylogenetic tree of the type shown in Fig 2. In this diagram, the branch lengths of the tree are related to the percent differences between the RNA

Bacteria Archaea E~,arya

Anier~=

sulphtl¢ INbcteri=

Pyrodictium

Cyanobactln=

T

Figure 2 Rooted phylogenetic tree constructed from 16S (or 18S) rRNA sequences. Estimates of sequence divergence (mutations fixed per sequence position) were calculated and used by Woese 4 to infer the tree

molecules of the various organisms being compared. The molecules which are used to construct these trees - - usually rRNA or, increasingly often, protein molecules - - are known as molecular clocks or phylogenetic markers.

The Application of Sequence Alignments to the Enzymes of Glycolysis Studies on the 3-phosphogiycerate kinases (PGK) of methano- genic archaea have shown these proteins to be 30-36% identical to their bacterial and eukaryal counterparts. 7 It is probable, therefore, that PGKs are all descended from a common ancestor. This is complemented by a preliminary study of the pyruvate kinase (PYK) of a thermophilic archaeum s which also shows considerable identity to regions of other PYKs. These data suggest a phylogenetic tree similar to that shown in Fig 2. In contrast, it appears that the archaeal glyceraldehyde-3-phos- phate dehydrogenases (GAPDH) are not homologous with

o 9 other GAPDHs as they exhibit only 15 '/o identity to them. This suggests that the archaeal GAPDH is descended from an entirely different protein than that which gave rise to the bacterial/

beta V H T E E S A L G K N V D V G R L L V V Y

beta Q R F E S T P D A V M N P K A G K K L G F S D G L L

beta N L K G T F A T E C D H E R G N V V C V H F

beta G K P A Y Q V V A N A A H H

alpha [~ L S P A D K T N V K A [ ~ W G K V beta H L T P E E K S A V L W G K

alpha T Y F P H F ~ H G S ~ Q V K G beta F F E S F G T P V M G N

alpha S A L S D L H A H K [ ~ R V ~ P V N beta L K G T F A T L S E H C K L H

alpha V H A S L D K F L A S V S T V L T beta E F T P P V Q A A Y Q K V V A G V

OA.AOE AE L E L SF V N V D E V G E A G R L L V V Y P Q R

G H K ~ V A D A L T N A V A H V D D M P N A L P K V A H G K K V L G A F S D G L A H L D N

F K L L S H C ~ ] V T L A A H L P ~ E F T P A V D P E N F R G N V L V C V L H H F G K

S K Y R A N A L A H K Y H

Figure 1 Alignments era- and G-haemoglobins with introduced gaps (upper panel) or with direct sequence alignment without gaps (lower panel). The gaps in the upper panel were inserted at three places (arrows) to maintain obviously similar sequences in register

B I O C H E M I C A L E D U C A T I O N 21(1) 1993

eukaryal GAPDHs. It is, therefore, possible that glycolysis arose as an association of enzymes which worked on substrates of similar structure. Some of the enzymes therefore share common ancestors and some of which are descended from different 'parent ' molecules. This information was recently exploited by the authors as a basis for an examination problem for final year undergraduate students (see sample problem section).

Glycolysis in the Thermophilic Arehaea Another valuable insight into the origins of glycolysis has come from the discovery that this pathway is not ubiquitous as had once been thought. In fact, in some thermophilic archaea, only two of the common ten enzymes are present, enolase (ENO) and PYK.t° This may suggest that the glycolytic pathway may have evolved from the 'bottom up' (ie in the gluconeogenic sense) starting with ENO and PYK, the only two parts of the pathway still thought to be ubiquitous. As time progressed, other enzymes joined the pathway some of which became common to all organisms. The final event in the assembly of the eukaryal glycolytic pathway was presumably the incorporation of 6- phosphofructokinase, the point at which bacterial glycolysis is blocked. Some of the thermophilic archaea seem to have been left behind by this process and hence may provide a glimpse into the distant evolutionary past of glycolysis.

Glycolysis may not be the only pathway to have its distant origins in thermophilic archaea. Archaeglobus fulgidus, a sulph- ate reducer, has recently been shown to possess elements of the methanogenerative pathway which was once thought to be unique to the methanogens.]l In conclusion, our opinions about the evolution metabolic pathways are still largely based on speculation but, with the recognition of the archaea and their unusual biochemistry, a powerful window onto the past has been opened by the molecular evolutionist.

Sample Problem for a Molecular Evolution Examination The following problem is based on work by Hensel et al 9 on the G A P D H enzymes of archaea and their relatedness to GAPDHs from organisms from the other two domains. The students were first given general information about G A P D H which catalyses the oxidative phosphorylation of glyceraldehyde 3-phosphate to glycerate 1,3-bisphosphate. In most organisms the enzyme has four identical subunits each of 330-340 amino acid residues, and the cofactor required for the reaction is NAD +. The amino- terminal half of the enzyme is involved in cofactor binding and the carboxy-terminal half provides the residues directly involved in catalysis. The students were also provided with information about the archaeal GAPDH: an enzyme recently isolated from the archaeum Methanobacterium formicicum that catalyses the same reaction except that the preferred cofactor is NADP +. The enzyme is a homotetramer with 337 amino acid residues per subunit.

The students were provided with a sequence alignment of GAPDHs from five sources which had been produced with CLUSTAL. The sequences aligned were from Drosophila, human, yeast, Bacillus stearothermophilus and Methanobac- terium formicicum. The alignment is shown in Fig 3 (although students were given an alignment of only the carboxy-terminal halves of the enzymes, from the residue marked with the *). Given this alignment, the students were asked to: (a) construct a table (matrix) of percent amino acid residue differences from all pairwise comparisons of the sequences. (b) use the information from the matrix to construct a simple phylogenetic tree. The students were then asked to discuss the possible evolution- ary history of the enzyme and to assess the usefulness of G A P D H as a molecular clock.

A matrix of differences is shown in Table 1 and the phylogenetic tree constructed from it is shown in Fig 4. The central importance attached to the tree was that it should

47

I r.n

Iqy , . M S K I G I N G F G R I G R L V L R A A I D K G . A N V V A V N D P F I D V K Y M V Y L F K . F Hum M G K V K V G V N G F G R I G R L V T R A A F N S G K V D I V A I N D P F I DL N Y M V Y M F Q . ¥ YOa , . M V N V S V N 6 F G R I G R L V T R I A I S R K D I N L V A I N D P F I S T D Y A A Y M F K . Y BOc . M A V K V G I NG F G R I G R N V F R A A L K N P D f EV V A V N D . L T D A N T L A H L L K , Y Mfr . . M K S V G I NG Y G T I G K R V A D A V S A O D D M K I V G V T K R S P D F E A R M A V E K G Y

51 IW F~ O $ T H G R F K G T V A A E G G F L V V N 6 Q K I T V F $ E R D P A N I N W A $ A G A E Y I V E S T Hum D S T H G K F H G T V K A E N G K L V I N 6 N P f T T F Q E R D P S K I K W G D A f i A E T V V E S T Yoo D S T H G R F D G E V S H D K D H I I L N G K K V A V F N [ K D P A A L P W G K L f l V D V A I D S T Bac D S V H G R L D A E V S V N G N N L V V N G K E I I V K A E R D P E N L A W G E I G V D I V V E S T ~ r D L Y I S A P E R E N S F E E A G i K V T G T A E E L F E K . . . . . . . . . . . L O I V V D C T

101 1~ F~ G V F T T I D K A $ T H L K G G A K K V I I S A P S A D A P M, F V C G V N L D A Y K P , D M K V V Hum G V F T T M E K A G A H L Q G G A K R V I I S A P S A D A P M. F V M G V N H E K Y D N . S L K I I Yea G r F K E M D S A N K H ~ E A G A K K V V I T A P S G S A P M. Y V M G V N E E T Y T P . D Q K I V Bac G R F T K R E D A A K H L E A G A K K V I I S A P A K N E O I T I V M 6 V N Q D K Y D P K A H H V I Mfr P . . . . E G I G A K N K E G T Y E K M G L K A T F Q G G E K H D Q I G L S F N S F S N Y K D V I G

151 20O Fly SN . . . . A S C T T N C L A P L A K V I N f l N F E I VEG L M T T V H A T T A T Q K T V D G P S G Hum SN . . . . A S C T T N C L A P L A K V I H D N F f i l V E G L M T T V H A I T A T Q K T V D G P S 6 Yea SN . . . . A S C T T N C L A P L A K V I H N E F f i l K E G L M T T V H S M T A T Q K T V D G P S H Bzc SN . . . . A S C T T N C L A P F A K V L H E Q F G I V R G M M T T V H S Y T N D Q R I L O L P . H Mfr K D Y A R V V S C N T T G L C R T L N P I N D L C G I K K V R A V M V R R G A D P S Q V K K G P I N

201 250 Fly K L W R O G R G A A QNI I P A S T G A A K A V G K V I PA L N G K L T G M A F R V P T P N V S V V Hum K L W R D G R G A L Q N I I P A S T G A A K A V G K V I P E L N G K L T G M A F R V P T A N V S V V Yea K D W R G G R T A S GNI I P S S T G A A K A V G K V L P S L Q G K L T G M A F R V P T V D V $ V V Bac K D L R R A R A A A E S I I P T T T G A A K A V A L V L P E L K G K L N G M A M R V P T P N V S V V Mfr A I V P N P P T . . . . . V P S H H G P . . O V Q T V M Y D L N . , I T T M A L L V P T T L M H Q H

251 300 F~y D L T V R L G K G A S Y D E I K A K V Q E A A N G P L . , K f l l L G Y T D E E V V S T D F L S D T Hum D L T C R L E K P A K T D 0 1 K K V V K Q A $ E G P L . . . K G I L G Y T E H Q V V S S D F N S D T Yea D L T V N L A K E T S Y D E I K A A L K K A S E G S M . . . KGI L G Y T E D D V V S S D F L G D A Bac D L V A E L E K E V T V E E V N A A L K A A A E G E L . . . KGI L A Y S E E P L V S R D Y N G $ T Mfr N L M V E L E S S V 51 DDI K D K L N E T P R V L L L K A K E G L G S T A E F M E Y A K E L G R $

301 350

Fly H $ S V F D . . . . A K A G I S L N D K F V K L I S W Y D N E F G Y S N R V I O L I K Y M Q S K D . Hum H S S T F D . . . . A G A G I A L N D H F V K L I S W Y D N E F G Y S N R V V O L M A H M A S K E . Yea H S S I VO . . . . A A A G I Q L T P T F V K L V S W Y D N E F G Y S T R V V O L V E H V A K S A . Bac V S S T I D . . . . A L S T M V I DGK M V K V V S W Y D N E T G Y S H R V V D L A A Y I A S K G L Mfr R N D L F E P G V W E E S L N I V O G E L Y Y M Q A I H Q E $ D V V P E N V D A P R A M L E M E D N

30? 365 ny H u m

Yea B a ¢

Figure 3 Sequence alignment of glyceraldehyde 3-phosphate dehydrogenase enzymes. Fly (Drosophila), Hum (human), Yea (Saccharomyces cerevisiae), Bac (Bacillus stearothermophilus), Mfr (Methanobacterium formicicum). The alignment was pro- duced using the program CLUSTAL and the sequences were obtained from the PIR database using the UWGCG package

Table 1 Matrix of differences between GAPDHs. Key as Figure 3

Fly Hum Yea Bac Fly Hum 30 - - Yea 39 37 - - Bac 49 51 48 - - Mfr 86 89 89 88

Mfr

correctly reflect the grouping of the different organisms. Hence, an approximation of the tree shown in Fig 4, showing the yeast, Drosophila and human sequences clustered together, the Bacil- lus sequence separated by a short branch, and the arehaeal sequence separated by a much longer branch was all that was required from the students. In the essay part of the paper, the students were asked to deduce from their crude tree that the archaeal enzyme is probably unrelated to the enzymes from the

B I O C H E M I C A L E D U C A T I O N 21(1) 1993

48

Bacitlus

S, cerevisiae

hila

Mb. fornlicicum

Figure 4 Phylogenetic tree constructed using matrix o f sequence differences derived from G A P D H alignment. The tree was constructed manually using branch lengths related to the degree o f difference between the sequences. The angles between the branches are arbitrary, assigned simply to make the viewing o f the diagram easier

bacterial and eucaryal sources which are interrelated. From this observation, it is clear that G A P D H would not be a useful molecular clock for comparing organisms from all three domains although it could be used for bacterial and eucaryal trees.

References 1Fothergill-Gilmore, L A (1991) 'The evolution of RNA and proteins as biocatalysts,' in Fundamentals of Medical Cell Biology, by Bittar, E E (editor) Vol 1, Evolutionary biology, JAI Press, London, pp 163- 188

2FothergilI-Gilmore, L A and Michels, P A M (1992) 'Evolution of glycolysis,' Prog Biophys Mol Biol (in the press)

3Rivera, M C and Lake, J A (1992) Science 257, 74-76 4Woese, C R, Kandler, O and Wheelis, M L (1990) Proc NatlAcad Sci,

USA 87, 4576-4579 5Higgins, D G and Sharp, P M (1988) Gene 73,237-244 ~Doolittle, R F (1986) 'Of Urfs and Orfs: A Primer on How to Analyse Derived Amino Acid Sequences,' University Science Books, Mill Valley, California

7Fabry, S, Heppner, P, Dietmaier, W and Hensel, R (1990) Gene 91, 19-25

8 Potter, S and Fothergill-Gilmore, L A (1992) FEMS Microbiol Lett 94, 235-240

9Hensel, R, Zwickl, P, Fabry, S, Lang, J and Palm, P (1989) Can J Microbiol 35, 81-85

l°Danson, M J (1988) Adv Microb Physiol 29, 165-231 ix Moller-Zinkham, D et al (1989) Arch Microbiol 152,362-367

Book Reviews

1992 Supplement to Biochemistry

D o n a l d V o e t and Jud i th G Voe t . pp 73. John Wi ley and Sons , N e w Y o r k . 1992. $2.95/£1.95 0 - 4 7 1 - 5 7 9 4 4 - 0

Is it worth while to review a slim volume that costs only a nominal sum? In the present case, the novel objective and unusual character of the book raise questions of general interest to teachers of biochemistry that merit analysis. The authors of a successful textbook are to be commended for seeking a novel solution to the problem of preventing their efforts from suffering

rapid obsolescence. I wish that I could conclude that they have succeeded.

The first question concerns the technical problem of integrat- ing new information so that readers are necessarily aware of additions and alterations. There might be several ways to approach this problem, including an index to the supplement, references to specific paragraphs in the original text, or a list of relationships. Since none of these exists, it is difficult to imagine how they might work and I am left with the feeling that students are not likely to integrate supplementary material easily as they use the text either as a primary source of information or as a reference.

In the introduction to the second supplement to their textbook, Donald and Judith Voet emphasize the importance of students and teachers keeping up with the literature. However, they do not make it clear whether the supplement is intended primarily for new students, who should be introduced to new information and recent articles as part of their first course in biochemistry, or whether it is intended for students who have completed their courses and are encouraged to update their texts so that the familiar book need not be replaced by more modern editions. Attempts by this reviewer to relate the supplementary material to the original text leave considerable doubt whether either purpose is served. Although I agree with the importance of students and practitioners of biochemistry surveying the literature regularly and familiarizing themselves with relevant items, I believe that the selection of relevant literature is highly personal and must be done individually as a function of professional interests. Therefore, my comments deal only with the supplement as a manual to be used together with the textbook during an introductory course in biochemistry.

It should be noted that the supplement contains brief discussions of information from current literature but that much of the supplement consists of mere references to review articles and primary papers. The 90 items selected for discussion average less than 3 per chapter; no additional text is given for 10 of the chapters and 11 of the items are related to the chapter on eukaryotic gene expression. In addition to this material, 199 references are listed as suggested reading for 25 of the 34 chapters. Do these references selected from perhaps 10 000 articles that appear each year in major biochemical journals represent basic information that would be likely to be included in a new text? The examples below suggest that the selection reflects the interests of the authors in structure-function relation- ships and that the items that have caught their fancies are presented in much greater detail than similar items are discussed in the original text.

Striking evidence of the personal interests of the authors is the fact that 40 of the 90 topics elaborated in the supplement are based on X-ray crystallography. There is no question of the value of this technology to elucidation of many problems of great current interest to active investigators in many areas of biochemistry. It would be a gross exaggeration, however, to maintain the enthusiasm that greeted David Phillips' study of lysozyme with the belief that the power of X-rays had made other aspects fo enzymology obsolete. One paper that is included deals with the fact that the core protein of the Sindbis virus is a serine protease. Since the virus is not described in either the original text or the supplement, the context of the information is obscure. It is also not clear why this serine protease is particularly worthy of addition to the 13 diverse members listed in the original text.

The only item presented as an advance in amino acid metabolism is an elaborate discussion of the mechanism by which chloroquine acts to kill the plasmodia that cause malaria. Although the studies do present new information in the way these organisms metabolize heine, it is doubtful that such a unique reaction is typical of the sort of information that should be included in any textbook.

B I O C H E M I C A L E D U C A T I O N 21(1) 1993