Redesign of Alpha Class Glutathione Transferases to Study ...161072/FULLTEXT01.pdf · Nilsson, L....

Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 672

_____________________________ _____________________________

Redesign of Alpha Class GlutathioneTransferases to Study Their

Catalytic Properties

BY

LISA O. NILSSON

ACTA UNIVERSITATIS UPSALIENSISUPPSALA 2001

Lisa O. Nilsson, Doctoral Thesis, 2001

Dissertation for the Degree of Doctor of Philosophy in Biochemistry presented at UppsalaUniversity in 2001

ABSTRACTNilsson, L. O. 2001. Redesign of Alpha Class Glutathione Transferases to Study TheirCatalytic Properties. Acta Univ. Ups. Comprehensive Summaries of Uppsala Dissertationsfrom the Faculty of Science and Technology 672. 30 pp. Uppsala. ISBN 91-554-5171-3.

A number of active site mutants of human Alpha class glutathione transferase A1-1(hGST A1-1) were made and characterized to determine the structural determinants foralkenal activity. The choice of mutations was based on primary structure alignments ofhGST A1-1 and the Alpha class enzyme with the highest alkenal activity, hGST A4-4, fromthree different species and crystal structure comparisons between the human enzymes. Theresult was an enzyme with a 3000-fold change in substrate specificity for nonenal over 1-chloro-2,4-dinitrobenzene (CDNB).

The C-terminus of the Alpha class enzymes is an -helix that folds over the activesite upon substrate binding. The rate-determining step is product release, which isinfluenced by the movements of the C-terminus, thereby opening the active site.Phenylalanine 220, near the end of the C-terminus, forms an aromatic cluster with tyrosine9 and phenylalanine 10, positioning the -carbon of the cysteinyl moiety of glutathione.The effects of phenylalanine 220 mutations on the mobility of the C-terminus were studiedby the viscosity dependence of kcat and kcat/Km with glutathione and CDNB as the variedsubstrates.

The compatibility of slightly different subunit interfaces within the Alpha class hasbeen studied by heterodimerization between monomers from hGST A1-1 and hGST A4-4.The heterodimer was temperature sensitive, and rehybridized into homodimers at 40 oC.The heterodimers did not show strictly additive activities with alkenals and CDNB. Thisresult combined with further studies indicates that there are factors at the subunit interfaceinfluencing the catalytic properties of hGST A1-1.

Lisa O. Nilsson, Department of Biochemistry, Uppsala University, Box 576, SE-751 23Uppsala, Sweden

Lisa O. Nilsson 2001-10-19ISSN 1104-232XISBN 91-554-5171-3Printed in Sweden by Eklundshofs grafiska, Uppsala 2001


"In the case of well studied protein frameworks where new enzymes can be designed andmade by small changes in residues close to the protein surface, we may hope it will soon beless laborious to redesign an existing and thermally stable framework for a new targetsubstrate than to search for a new enzyme activity from natural organisms"

H. M. Wilks et al. (1988)


This thesis is based on the following publications, which will be referred to by their Romannumerals in the text:

I Nilsson, L. O., Gustafsson, A. and Mannervik, B.Redesign of substrate-selectivity determining modules of glutathione transferaseA1-1 installs high catalytic efficiency with toxic alkenal products of lipidperoxidation. Proceedings of the National Academy of Sciences U. S. A. (2000) 97, 9408-9412

II Nilsson, L. O., Etahadieh, M., Pettersson, P. and Mannervik, B.Aromatic residues in the dynamic C-terminal region of glutathione transferase A1-1 influence rate-determining steps in the catalytic mechanism.submitted

III Nilsson, L. O. and Mannervik, B.Improved heterologous expression of human glutathione transferase A4-4 byrandom silent mutagenesis of codons in the 5' region.Biochimica et Biophysica Acta (2001) in press.

IV Gustafsson, A., Nilsson, L. O. and Mannervik, B.Hybridization of Alpha class subunits generating a functional glutathionetransferase A1-4 heterodimer. submitted

Reprints were made with permission from the publishers.


CONTENTS

BACKGROUND 1

ENZYMES, THE CATALYSTS OF NATURE 1A possible strategy for enzyme reconstruction 3

GLUTATHIONE TRANSFERASES 10Development of different catalytic machineries 13

PRESENT WORK 15

OBTAINING HIGH EXPRESSION LEVELS OF PROTEINS (PAPER III) 15

ROLE OF RESIDUE PHE 220 IN THE POSITIONING OF THE C-TERMINUS (PAPER II) 16

SPECIFICITY DETERMINING REGIONS WITHIN THE ACTIVE SITE OF ALPHA CLASSGLUTATHIONE TRANSFERASES 16

Catalytically important differences in the H-site between glutathione transferase A1-1and glutathione transferase A4-4 (paper I) 19

INFLUENCE OF THE SUBUNIT INTERFACE ON THE CATALYTIC PROPERTIES (PAPER IV) 21

CONCLUDING REMARKS 22

ACKNOWLEDGEMENTS 23

REFERENCES 25

BACKGROUND

Lisa O. Nilsson, Doctoral Thesis, 2001 1

BACKGROUND

ENZYMES, THE CATALYSTS OF NATURE

What makes it possible to coordinate all the reactions necessary for a living organism tofunction? How can everything work smoothly without accumulation of intermediates in thereaction pathways, buildup of byproducts, and with every function only turned on whenneeded? All reactions in the body would be slow and will not work without the help of acatalyst, i. e. something that increases the rate of reaction without being consumed in theprocess. This makes it possible to regulate the reactions by turning the catalyst on and off.A catalyst can be a surface that adsorbs the reacting molecules, substrates, making it easierfor them to find each other. It can also be a molecule that stabilizes polar reactionintermediates. A catalyst can enhance the rate of reaction by changing the reactionpathway. An example is when it is impossible to make two molecules react with each otherdirectly. Then an intermediate step can be introduced generating a good leaving group,which makes it easier for the second substrate to react thereby regenerating the catalyst.

Nature has taken care of catalysis in a way that is still not fully understood. She has builtlarge molecules, enzymes, with a cleft where the reacting molecules, substrates, can meet.Only one or a range of specific molecules can bind depending on the shape of the cleft,active site, and only if hydrophobic and hydrophilic groups on the substrate molecules canbe accommodated. Through analysis of a crystal structure it is fairly easy to identifypotential catalytic groups in the active site and determine residues that contact the substrate.Mutagenesis is a method that can be used to verify the roles of those residues. Elucidatingthe role in catalysis of the rest of the huge molecule has so far proved very difficult.

REDESIGN OF ALPHA CLASS GLUTATHIONE TRANSFERASESTO STUDY THEIR CATALYTIC PROPERTIES

2 Lisa O. Nilsson, Doctoral Thesis, 2001

Enzymes are among the most efficient catalysts known. They can be very specific for onesubstrate, thereby limiting the byproducts, and they can enhance the rate of a reaction untilit is diffusion limited, i. e. 109 s-1M-1 (Fersht, 1999). Few non-enzymatic catalysts knowncan compete in the mild aqueous conditions where enzymes operate. It would be veryuseful if it were possible to synthesize enzymes that could work as catalysts for industrialsynthesis of desired products such as pharmaceuticals. In many cases molecules can reactwith each other in several different ways yielding many different products. When makingsucrose, for example, one hydroxyl group on glucose has to react with one hydroxyl onfructose. A conventional catalyst would enhance the rate of reaction between any of thehydroxyl groups on the sugars (Fig. 1).

Figure 1. D-glucose and D-fructose gives sucrose.

Therefore all the hydroxyl groups, but the ones taking part in the reaction, have to beprotected. In other words the synthesis has to be carried out in several steps. Each step willgive byproducts, thereby lowering the yield of the final product. If it was possible to use anenzyme as a catalyst instead, the sugars would bind to the active site in a specific wayleaving only one hydroxyl group free to react, thus reducing the reaction pathway to onestep. Formation of byproducts is minimized since the rate of the side-reactions are not asenhanced by the catalyst. There are many benefits to enzyme catalysis. There is nounnecessary waste of starting-material and purification of the product is superfluous as theenzymatically driven reaction is so much faster than the uncatalyzed one. The production issimplified, thereby reducing the cost. There are products impossible to synthesize today.By changing the reaction pathway enzymes could make it possible.

O

CH2OH

OH

OH OH

OHO

OH

OH

CH2OH

CH2OH

+ -H20 O

OH

OH

CH2OH

CH2OH

O

O

CH2OH

OH

OH

OH

BACKGROUND


So far science has been successful in customizing existing enzymes. In washing powder,for example, there are protein cleaving enzymes, proteases, which help remove proteinstains.

Unfortunately there are not natural enzymes for every reaction we wish to catalyze.Therefore several different approaches are used in the struggle to custom design enzymes.They can be divided into two major groups, random and rational methods. Randommethods usually involve making a library of a vast number of different mutants, which canthen be screened for a certain property (Dunn, 1996, Altamirano et al., 2000). However, itis hard to know what mutations to introduce into the library and to find a screening methodthat is specific enough. Rational methods usually involve site-directed mutagenesis, or therecombination of different parts of proteins, domains, based on knowledge extracted fromknown crystal structures and sequence alignments. In the present investigation onlyrational methods have been used.

A possible strategy for enzyme reconstruction One way of looking at enzymes is that they serve as a scaffold for the active site. Thisimplies that the reaction of an enzyme can be changed, by replacing its active site with adifferent one.

There have been many attempts to use existing proteins as scaffolds for different activesites, where only the target substrate or the whole mechanism is changed. A lot ofinformation about determinants of enzyme specificity has been accumulated along the way,but it is still not straightforward to tailor functions into existing scaffolds. The role of thebulk part of the enzyme in catalysis, is still a mystery. Random insertions of mutations inparts of the enzyme remote from the active site elevate enzyme activity significantly.However, the reason for this is still not well understood.



Neet et al. (1966) and Polgár et al. (1966) are considered to be the first to alter an enzymethrough rational modifications. They tried to improve the activity of the serine proteasesubtilisin (from Bacillus subtilis) by chemically modifying the active site serine intocysteine. The rationale behind the attempt was that since cysteine is more nucleophilic thanserine it would make a better catalyst. Cysteine proteases are also found in nature, oneexample being papain (from papaya). Unfortunately, the modification effectively killedsubtilisin. The active site serine in the stomach protease trypsin was also replaced with acysteine rendering the same result (Yokosawa et al., 1977). The lesson is that enzymes usea delicately fine-tuned set of steps in order to carry out their reactions at high speed. Forexample, the nucleophile used can not be too strong because then it will make a poorleaving group and the enzyme will not be able to release the product and regenerate itself.

Another approach with a lot of practical significance is semisynthesis, where shortersequences in a protein are replaced by chemically synthesized ones. This results in achimeric protein, i. e. a protein with parts from different sources (Offord, 1987).Semisynthesis has been used for humanisation of porcine insulin (a hormone) (Markussen,1982). The only differences between human and porcine insulin are found in the last eightresidues of one of the two peptide-chains that make up the protein. This part was cut offand replaced by a chemically synthesized human equivalent. In this way side effects fromtreating patients with porcine insulin could be avoided (Bliss, 1993). Today more efficienttechniques are used.

Semisynthetic enzymes have also been made by covalently binding coenzymes to an activesite (Kaiser, 1988). The resulting hybrid enzymes couple the unique chemistry of thecofactor with the binding specificity of the protein template. Kaiser and coworkers haveconstructed semisynthetic flavoenzymes by incorporating reactive flavin analogs into theactive site of papain (Slama et al., 1984) and glyceraldehyde-3-phosphate dehydrogenase(Hilvert et al., 1988).

BACKGROUND


In 1989 redesign of subtilisin and trypsin came into focus again. Wu and Hilvertsuccessfully converted subtilisin into an acyl transferase by conversion of the active siteserine into selenocysteine (Wu et al., 1989, Wu et al., 1990). Higaki et al. (1989) reasonedthat chemical modification of enzymes could give side-effects rendering the enzyme dead.Therefore, they tried to reconstruct the putative catalytic triad of the cysteine proteasepapain within the serine protease trypsin. This time using a different method, site-directedmutagenesis. Still, no activity was observed. A crystal structure was generated for themutant (McGrath et al., 1989). The conclusion was made that the position of the thiolappears to obstruct the structural feature responsible for the stabilization of the oxyanionformed during the reaction. Hilvert (1991) has reviewed how selective chemicalmodifications in vitro can be used successfully to create novel proteins with alteredspecificities and catalytic activities.

Wilks et al. (1988) were able to elucidate the determinants for substrate specificity ofmalate dehydrogenase and lactate dehydrogenase (LDH). The rate-limiting step was foundto be the structural rearrangement of the enzyme-NADH-substrate complex prior to thechemical step. This rearrangement is dominated by the closure of the active-site loop. Itturned out that one residue in this loop determined whether the loop could close over thesubstrate or not, thereby affecting the activity. The catalytic efficiency, kcat/Km , of nativeLDH and the mutant are presented in Table 1.

Further attempts to exchange substrate specificities between enzymes with similarfunctionalities by rational design have been carried out with varying degrees of success.Even if it has proved possible to change the substrate specificity, it is rare for mutantenzymes to become as efficient as naturally occurring ones in terms of catalytic efficiency(kcat/Km). Examples of successful studies are presented in Table 1.

An early redesign experiment changed the coenzyme specificity of glutathione reductase. Ittook seven mutations to alter its preference for coenzyme NADH over NADPH (Scrutton etal., 1990). Essentially, this meant filling up the pocket in the active site where thephosphate on NADPH was supposed to bind. Chen et al. (1995) tried to do it the other wayaround, i. e. create a pocket for the phosphate of NADPH in wild-type Escherichia coliisocitrate dehydrogenase. Seven amino acid substitutions were also needed in this case(Table 1).



Trypsin and chymotrypsin are two serine proteases that cleave peptide bonds adjacent tolong charged residues (arginine and lysine) and aromatic residues (phenylalanine, tyrosineand tryptophan), respectively. The obvious determinants of their substrate specificities arefound in the binding pocket where the amino acid residue, adjacent to the peptide bond tobe cleaved, is bound (Fig. 2).

Figure 2. The binding pockets oftrypsin (left) and chymotrypsin(right). The pocket where theamino acid residue adjacent to thepeptide bond to be cleaved isbound.

The shapes of the pockets are essentially the same but in trypsin there is a negativelycharged aspartate at the bottom to neutralize the charge of the arginine or lysine residue.The binding pocket of chymotrypsin is hydrophobic to accomodate the aromatic rings oftheir preferred substrate. It seemed like it would be fairly easy to exchange their activities.However, when attempted by Hedstrom et al. (1992) it turned out that the binding pocketdetermines the specificity of ester hydrolysis, with substrates tried. Whereas specific amidehydrolysis requires more distant binding site interactions in addition to binding pocketinteractions. The distant binding sites involve two surface loops that connect the walls ofthe binding pocket without direct substrate contact. The loops were targeted by sequencealignment. The catalytic efficiencies of the mutant and the wild type enzymes are presentedtable 1.

BACKGROUND


Introduction of mutations into the active site of an enzyme usually decreases its originalcatalytic activity. However, when Onuffer et al. (1995) succeeded in introducingEscherichia coli tyrosine aminotransferase (eTATase) activity into Escherichia coliaspartate aminotransferase (eAATase) the original activity with aspartate was retained. Theobvious target for mutagenesis this time seemed to be a charged arginine residue at thebottom of the substrate-binding pocket that neutralizes the aspartate. Replacement of thearginine with a hydrophobic residue was expected to favor tyrosine binding. This proved tobe unnecessary. Instead six residues close to the active site found to be conserved in manyaspartate aminotransferase enzymes but not in tyrosine aminotransferases were mutated.The resulting enzyme had increased activity with phenylalanine (which was used instead oftyrosine), without sacrificing the high transamination activity with aspartate. The mutantafforded this dual specificity by letting the charged amino acid flip-flop in and out of theactive site depending on the substrate bound.

There is an interest in making transgenic oilseed crop plants producing specific fatty acids.Some potential uses include the production of low-caloric margarine and as a precursor inthe manufacture of nylon. Also, the quality of unsaturated fatty acids in vegetable oilscould be enhanced. Therefore, Cahoon et al. (1997) set out to find the determinants forsubstrate specificity and the site where double bonds are introduced (see Table 1 for theresults).

One of the few examples where it was actually sufficient to change only one amino acidresidue in order to introduce a different substrate specificity is given by Jez et al. (1998).Guided by the three-dimensional structure of rat liver 3 -hydroxysteroid dehydrogenase(3 -HSD) and sequence comparisons of steroid-metabolizing aldo-keto reductases (AKRs),5 -reductase activity was successfully engineered into 3 -HSD by means of a single pointmutation (Table 1).



Table 1. Examples of successful introductions of new substrate specificities by rational redesign. kcat/Km is beingcompared unless otherwise stated.

enzyme substrate 1 substrate 2 specificity = kcat/Km substrate 1 kcat/Km substrate 2

chloro-2,4-dinitro- nonenalbenzene

GST A1-1 a) 160 mM-1s-1 5.0 mM-1s-1 32

GIMFhelix a) 14 mM-1s-1 1520 mM-1s-1 0.009

GST A4-4 a) 7.9 mM-1s-1 485 mM-1s-1 0.016

pyruvate oxaloacetate

LDH native b) 4.2x106 M-1s-1 4.0x103 M-1s-1 103

LDH Gln102Arg b) 5.0x102 M-1s-1 4.2x106 M-1s-1 10-4

NADPH NADH

glutathione reductase c) 740.0 min-1/µM-1 0.34 min-1/µM-1 2x103

mutant (7mutations) c) 3.0 min-1/µM-1 24.4 min-1/µM-1 10-1

AAP-Phe-AMC AAP-Lys-AMC

trypsin d) 4.5 M-1s-1 1.2x106 M-1s-1 10-6 to 10-7

trypsin mutant d) 2.8x103 M-1s-1 34 M-1s-1 102 to 103

chymotrypsin d) 1.6x106 M-1s-1 850 M-1s-1 102 to 103

kf/Kd aspartate kf/Kd phenylalanine

eAATase e) 200 x10-3 M-1s-1 0.25 x10-3 M-1s-1 800

eAATase mutant (6 mutations) e) 340 x10-3 M-1s-1 370 x10-3 M-1s-1 0.9

eTATase e) 54 x10-3 M-1s-1 2300 x10-3 M-1s-1 0.02

BACKGROUND


Table 1 (continued)

NADP NAD

isocitrate dehydrogenase (ICDH) f) 4.7µM-1s-1 0.69x10-3 µM-1s-1 6900

ICDH mutant f) 0.81x10-3 µM-1s-1 0.164 µM-1s-1 0.005

isopropylmalat dehydrogenase f) 2.4 x10-3 µM-1s-1 0.34 µM-1s-1 0.007

testosterone 5 -androstan-

17 -ol-3-one

3 -HSD g) ND 9500 min-1/mM-1 0:9500

3 -HSD H117E g) 13 min-1/mM-1 9.4 min-1/mM-1 1.4

5 -reductase g) 565 min-1/mM-1 blank 565:0

Specific activities (nmol/min/mg protein)16:0-ACP 18:0-ACP

6 9 6 9

6-16:0-ACP desaturase h) 100 ND 11 5.5 6

mutant 1 h) 227 ND 253 5.3 1

mutant 2 h) 19 1.4 ND 76 0.39 16:0-ACP 9 18:0-ACP

9-18:0-ACP desaturase h) ~0.2 ~9.7 0.02

mutant 3 h) ~2.8 ~1.2 2

ND, not detectable, a) paper I, b) Wilks et al. (1988), c) Scrutton et al. (1990), d) Hedstrom et al. (1992), e)

Onuffer et al. (1995), f) Chen et al. (1995), g) Jez et al. (1998), h) Cahoon et al. (1997)

A comprehensive summary of recent strategies in the rational redesign of enzyme substratespecificity and mechanisms has been presented elsewhere (Cedrone et al., 2000).

Site-directed mutagenesis, phage display, chemical modification, and other tools for proteinengineering are now used routinely both to redesign proteins and to answer specificbiological questions. Remarkable results in the field of protein redesign have been achievedwhen looking for something else (Doyle et al., 1998). When using these methods vitalinformation from an experiment may be overlooked unless experts in both proteinengineering and on the system being studied are engaged (Doyle et al., 1998). This thesis is



an example of how redesign can be used to gain information about the enzyme being used,besides providing a tool for the customization of enzymes.

The examples above show that it is not a simple task to predict which amino acidsubstitutions are going to be crucial when introducing novel activities, although there arerational ways to go about it. As can be seen in Table 1, the specificity of the new enzymesare successfully changed, but in most cases the catalytic efficiencies obtained are poorcompared to the parental enzymes. Usually the attempts to exchange substrate specificitiesdo not involve a change in reaction mechanism.

GLUTATHIONE TRANSFERASES

All living organisms need to protect themselves from toxic molecules which either arebyproducts of reactions within the body, or enter from outside through the skin, the lungsor with the food we eat. Only water-soluble molecules can be easily excreted from the bodythrough sweat and urine. In contrast, hydrophobic molecules may be dissolved inmembranes throughout the body and disturb their functions. Therefore, the body needs away to turn hydrophobic molecules into hydrophilic ones. The specific enzymes with thistask are called Phase I enzymes. Unfortunately the result of Phase I enzymes is sometimesdevastating to the cell. In some cases epoxides are formed and they react readily with anynucleophile nearby, such as DNA and proteins. Phase II enzymes disarm these veryelectrophilic molecules. The glutathione transferases belong to the Phase II enzymes andthey make the toxic products react with glutathione, rendering them harmless. Glutathione(GSH) is a highly charged tripeptide (Fig. 3). The products are thus more water-soluble.

Figure 3. Glutathione, -Glu-Cys-Gly

O NH

NH

O

NH3+

OSH

O

O

O

BACKGROUND


The following criteria need to be met by glutathione transferases in their role asdetoxication enzymes:- a capacity to bind a broad range of substrates - a machinery with which to catalyze several different types of reactions (see below)

Glutathione transferases are thought to have other functions in the body as well, such astaking part in metabolism and as a transporter protein. Some examples that illustrate thebroad range of reactions that are efficiently catalyzed by glutathione transferases arepresented below.

– Addition reactions, with -unsaturated compounds

– Aromatic substitution reactions

ClNO2

NO2

GS-NO2

NO2

SG

+Cl-

ROH

OH

GS- H+

ROH

GSH

H

O



– Reduction of hydroperoxides

– Reduction of epoxides

– Isomerization of steroids

O

O

O

O

RO O

H

2GSH

ROH

GSSG

H2O+

O

R R'

GS-H+

R R'

OHGS

BACKGROUND


Development of different catalytic machineries Different classes of soluble glutathione transferases with unique substrate profiles haveevolved in order to accommodate as many different substrates as possible. The followingclasses have been identified so far in humans: Alpha, Kapa, Mu, Omega, Pi, Sigma, Thetaand Zeta. They differ in primary, secondary and tertiary structure.

Glutathione transferases are dimers and can be either homodimeric, i. e. consists of twoidentical subunits, or heterodimeric, i. e. consists of two different subunits.Heterodimerization increases the variability of the system, as different functions can bebrought close together. Heterodimers can only form between members within the sameclass. The subunits often operate as independent units only using each other forstabilization of their structures (Danielson et al., 1985, Tahir et al., 1986, Gustafsson et al.,1999). However, now evidence indicating communication between the subunits in ratglutathione transferase 1-1 (Wang et al., 2000) and mouse glutathione transferase A4-4 isgathering (Xiao et al., 1999).

Each subunit contains two domains where the N-terminal is dominated by -sheets and theC-terminal is made up of -helices (Fig. 4). The active site is divided into two bindingpockets called the G-site, where glutathione binds, and the H-site, for the hydrophobicsubstrate. The G-site is located in the N-terminal domain and the H-site is in the C-terminalone. The fold of the polypeptide chain of the glutathione transferases is the same within aclass, and to a great extent between classes. However, the detailed structures of the H-sitesdiffer from one another.



A: Alpha B: Mu C: Omega

D: Pi E: Theta

Figure 4. The crystal structures of one subunit of human glutathione transferases (GSTs) from fivedifferent classes. A: GST A1-1 (Sinning et al., 1993), B: GST M2-2 (Raghunathan et al., 1994), C: GST O1-1(Board et al., 2000), D: GST P1-1 (Reinemer et al., 1992), E: GST T2-2 (Rossjohn et al., 1998). The C-terminus,which is part of the H-site, is highlighted in black. The subunits are divided into two structurally independentparts, domains. One to the left in the pictured with mainly -sheets and one to the right with -helices.

The H-site in the Alpha class enzymes is made up of the following three regions (Fig. 7): – the 1- 1 loop, residues 9-16, – part of the 4 helix, residues 101-111,– the C-terminus, residues 208-222. In this thesis it is shown that changes in these regions can be sufficient to alter the substratespecificity and the mechanism of Alpha class glutathione transferases.

PRESENT WORK


PRESENT WORK

OBTAINING HIGH EXPRESSION LEVELS OF PROTEINS (PAPER III)Some characterization methods require large amounts of protein. In order to be able tocollect enough protein in a reasonable amount of time, it is essential to have as highexpression level as possible. The method used to make heterodimers between differentsubunits required that they could be coexpressed in approximately equal amounts. Aheterodimer between glutathione transferase A1-1 and A4-4 has been studied (paper IV).The expression level of glutathione transferase A4-4 was too low for heterodimerization tooccur, therefore a new expression clone was made.

It has been shown that the following factors affect the level of expression: – codon usage, because organisms are biased in their use of codons (Andersson et al.,1990). The expression level seems to be most sensitive to the codons in the 5'-end. – availability of tRNA (Ikemura, 1981b, Ikemura, 1981a) affects the rate of translationinitiation (Bergmann et al., 1979). – codon-anticodon binding energy (Grosjean et al., 1978, Fiers et al., 1979, Grantham etal., 1981, Gouy et al., 1982). A cluster of G and C at the 5'-end can make is difficult for thepolymerase to denature the DNA, thereby limiting expression.– secondary structure formation in the mRNA, that can cause the ribosome to fall off.

It is not possible to predict the effect of each of these factors on a specific codon.Therefore, a random approach to elevate protein expression was used. A library of silentmutations in the first 10 codons in the 5'-end was constructed. The library was screened forthe clone with highest expression level by dot blot analysis (paper III). The identified cloneelevated the expression level 2-10 fold depending on the culture volume. Smaller volumesgives more enzyme/liter cell culture. The identified clone made it possible to make aheterodimer between glutathione transferase A1-1 and A4-4 by coexpression (paper IV).



ROLE OF RESIDUE PHE 220 IN THE POSITIONING OF THE C-TERMINUS(PAPER II)Analogous to malate dehydrogenase and lactate dehydrogenase, the rate-limiting step ofAlpha class glutathione transferases with most substrates is product release. Due to astructural rearrangement dominated by the movement of an active-site sequence, in thiscase the C-terminus. The closure of the helix blocks out water from the active site, resultingin a higher specificity towards hydrophobic substrates. Few specific van der Waals contactsbetween the C-terminal residues and the hydrophobic ligand are observable. Therefore, ithas not been possible to elucidate the basis for the ligand-driven structural rearrangementof the C-terminus merely by studying the crystal structure.

There are three residues, tyrosine 9, phenylalanine 10 and phenylalanine 220 that form anaromatic cluster that aid in the positioning of the -carbon of the cysteinyl moiety ofglutathione (Cameron et al., 1995). Nieslanik et al. (2000) have studied this cluster in ratglutathione transferase A1-1. They speculate that the changes in the hydrogen bondstrength of tyrosine 9 alter the electrostatic surface of its aromatic ring, thus altering theinteraction between the electropositive edge of phenylalanine 10 in the open state and theedge of phenylalanine 220 in the closed state.

Since the rate-determining step in the catalytic cycle of glutathione transferase A1-1 isproduct release, probably due to the rearrangement of the C-terminus, the rate constant kcatis expected to be sensitive to the viscosity of the buffer used during the assays. This wasindeed the case (paper II). The magnitude of kcat/Km is also affected by viscosogen,indicating that glutathione transferse A1-1 is diffusion controled (paper II). By mutatingphenylalanine 220 into an alanine, the positioning of the C-terminal sequence uponsubstrate binding was impaired, rendering kcat and kcat/Km insensitive to the viscosity of thebuffer. A double mutant, where both phenylalanine 220 and 222 were replaced by alanines,behaved in essentially the same way (paper II). Only the phenylalanine 220 mutation isrequired to make the enzyme insensitive to viscosogen, indicating that it is important forthe substrate induced positioning of the C-terminus over the active site.

SPECIFICITY DETERMINING REGIONS WITHIN THE ACTIVE SITE OF ALPHACLASS GLUTATHIONE TRANSFERASES (PAPER I)Since the major differences between glutathione transferases within a class are found in andaround the H-site of the enzymes, it has been tempting to draw the conclusion that thesubstrate specificity can be altered by changes in residues in this region only. In a previousstudy by Hansson et al. (1999) an attempt was made to introduce glutathione transferase

PRESENT WORK


M2-2 activity into glutathione transferase M1-1 using both rational and randomapproaches: site-directed mutagenesis and DNA shuffling. The enzymes share 84 %sequence identity and show a 100-fold difference in activity with the glutathionetransferase M2-2 characteristic substrates aminochrome, 2-cyano-1,3-dimethyl-1-nitrosoguanidine and 1,2-dichloro-4-nitrobenzene. It was demonstrated that two regions inthe C-terminal domain and parts of the 4 and 6 helices were important for substrateselectivity. The best glutathione transferase M1-1 mutant lacked one order of magnitude ofthe activities with the characteristic substrates of glutathione transferase M2-2. As in earlierstudies with other enzymes (Table 1), it was concluded that unpredictable second sphereinteractions played a great role in determining the catalytic properties.

In the present work (paper I) rational design was used to elucidate the determinants forsubstrate specificity in the Alpha class. Two of the enzymes, glutathione transferase A1-1and glutathione transferase A4-4 have 53% identical primary structure and their proteinfolds are essentially the same (Fig. 5). However, their substrate selectivities are distinctlydifferent (Hubatsch et al., 1998).

Figure 5. Crystal structures of glutathione transferases A1-1 (left) and A4-4 (right). No significant distinctioncan be made between the two enzymes without looking at the primary structure. The structures were determinedby Sinning et al. (1993) and Bruns et al. (1999).



Glutathione transferase A1-1 readily catalyzes aromatic substitution (Fig. 6). This meansthat it has to break aromaticity, change configuration around a carbon from sp2 to sp3, andaccommodate the deformation of the ring of the electrophilic substrate. Glutathionetransferase A4-4 preferably catalyzes Michael addition to alkenals (Fig. 6). This means thatit has to polarize the carbonyl, break a double bond, donate a proton, H+, and orient theelongated substrate in a productive way inside the active site.

Figure 6. Substrates used to discriminate between glutathione transferase A1-1 and A4-4.

PRESENT WORK


Catalytically important differences in the H-site between glutathionetransferase A1-1 and glutathione transferase A4-4 (paper I)The H-sites of the two enzymes have different shapes. Glutathione transferase A1-1 has abroad and shallow site, which can accommodate aromatic rings. Whereas the site ofglutathione transferase A4-4 is long and narrow and can accommodate alkenals. The H-siteis situated between the C-terminus and the 1- 1 loop, with part of the 4 helix making upthe bottom (Fig. 7).

Figure 7. The active site of glutathione transferase A1-1 (left) and A4-4 (right). The picture have been rotated90o compared to Fig. 4A. Bound ligand is colored black (S-benzyl-glutathione and S-o-iodobenzyl-glutathione,respectively). The amino acid residues in the 9 helix (in the C-terminus), pointing into the active-site, are bulkierin glutathione transferase A4-4 creating a narrower cleft. The residues 107, 108 and 111 at the bottom of the H-site are oriented towards the ligand in glutathione transferse A1-1, giving a shallower cleft, whereas in glutathionetransferase A4-4 they are oriented away from the ligand, making room for the slim substrates.

Previously several amino acid residues have been identified as important in the catalysis ofalkenals. In this study, however (paper I), glutathione transferase A4-4 specific activitieshave been introduced into glutathione transferase A1-1 by rational redesign (paper I). Thisincludes a change both in substrate selectivity and in the mechanism of reaction. Theresidues to be modified were identified by structure comparisons and sequence alignmentsincluding rat, mouse and human DNA. 14 amino acid residues were targeted. The resultwas an enzyme with a 3000-fold change in substrate specificity for nonenal over 1-chloro-2,4-dinitrobenzene (CDNB). This is the first time the resulting enzyme is as efficient as its



wild-type counterpart (Table 1). From this study the specificity determining factors couldbe deduced and they are presented below.

The different shapes of the active sites are mainly due to differences in the C-terminus andto three amino acid residues in the 4 helix. In glutathione transferase A1-1, the C-terminushas small hydrophobic amino acid residues pointing into the active site. In glutathionetransferase A4-4 the corresponding residues are bulky. This might also make the C-terminal helix more rigid (Bruns et al., 1999). The structural differences are clearly visiblein the crystal structures (Fig. 7). In the glutathione transferase A1-1 apoenzyme, i. e.enzyme without bound substrate, the C-terminus is not visible, which means that it ishighly mobile (Cameron et al., 1995). In contrast the C-terminal sequence of glutathionetransferase A4-4 is visible but for the last two residues 221 and 222 (Bruns et al., 1999).

At the base of the C-terminal helix (position 208) there is a methionine in glutathionetransferase A1-1 and a proline in glutathione transferase A4-4. As proline isconformationally restricted it could have an effect on the orientation of the whole C-terminus.

At the bottom of the cleft, glutathione transferase A1-1 contains leucines (positions 107 and108) and a valine (position 111) where glutathione transferase A4-4 contains isoleucine,methionine, and phenylalanine at the structurally homologous positions, respectively.Methionine 108 is oriented in such a way that a cleft is formed between isoleucine 107 andphenylalanine 111 (fig. 7), where the tail of the alkenal substrate can bind (Nanduri et al.,1999).

The reaction with alkenals includes the polarization of an aldehydic carbonyl. The protondonor in glutathione transferase A4-4 has been identified as a tyrosine in position 212 nearthe base of the C-terminal helix (Hubatsch et al., 1998). There is room for this residue inthe H-site only if there is a glycine in position 12 in the 1- 1 loop (Björnestedt et al.,1995). Glutathione transferase A1-1 does not have this need and therefore lacks thistyrosine.

In conclusion, glutathione transferases can recruit residues from different parts of theprimary structure to build different active sites at its convenience.

PRESENT WORK


INFLUENCE OF THE SUBUNIT INTERFACE ON CATALYTIC PROPERTIES(PAPER IV)Depending on the compatibility of subunit interfaces of different monomers, theirheterodimers form more or less readily. A system has been set up in which heterodimerscan be produced directly in Escherichia coli by heterologous coexpression (Gustafsson etal., 1999). When exposed to each other in such high quantities, as is the case when theiroverexpressed together in a cell, the equilibrium can be shifted toward heterodimerizationeven when it is unfavorable. This made it possible to study heterodimerization between lesscompatible subunits such as the monomers A1 and A4.

The heterodimer glutathione transferase A1-4 could be formed, only when the expressionlevels of both enzymes were sufficiently high. Once formed it was stable only at lowtemperatures. At 40 oC the two subunits dissociated and reassociated into homodimers. Animportant finding was that the activities with model substrates for each enzyme, CDNB andnonenal, were not strictly additive for the heterodimer (paper IV) as opposed to otherheterodimers studied previously (Danielson et al., 1985, Tahir et al., 1986, Gustafsson etal., 1999). It was not possible to deduce if the effect on the activities was caused bydestabilization of the enzyme by incompatible structure motifes at the subunit interface orsome form of negative cooperativity.

Recent studies have provided more obvious indications of communication between thesubunits. It has been reported that larger substrates bind to Alpha class enzymes with astoichiometry of 1 mol/(mol of enzyme dimer) (Schramm et al., 1984). Smaller substratesbinds with a stochiometry of 2 mol/(mol of enzyme dimer) (McHugh et al., 1996). Wang etal. (2000) demonstrated that rat glutathione transferase 1-1 can be inactivated with largesubstrates by specifically modifying the active site of only one subunit. When combiningthe modified subunit with an unmodified subunit from glutathione transferase 2-2 noinactivation of the unmodified subunit can be seen. Thus, this effect seems to be specificfor the glutathione transferase A1-1 enzyme.



Lien et al. (2001) have studied subunit interactions in human glutathione transferase A1-1using a heterodimer between an unmodified subunit and one largely inactivated by amutation in the active site. They clearly demonstrate that the glutathione transferases mayhave either half-of-the sites or all-of-the-sites reactivity depending on the nature of thesubstrate. Glutathione transferase A1-1 displays half-of-the-sites reactivity in the Michaeladdition of glutathione to nonenal. Evidence for half-of-the-sites reactivity has beenreported for mouse glutathione transferase A4-4 as well (Xiao et al., 1999). Although thestudy presented here was not conclusive, the studies mentioned above strongly indicate thatthere are factors at the subunit interface influencing the catalytic properties.

CONCLUDING REMARKS

The catalytic properties of Alpha class glutathione transferases are governed by both thestructure of specific regions in the active site and by interactions between the subunits.Mutations only in the substrate binding regions can change both substrate specificity andenzyme mechanism without compromising a high catalytic efficiency. The Alpha classglutathione transferases are suitable candidates for enzyme redesign studies concerningenzyme function and scaffold design.

ACKNOWLEDGEMENTS


ACKNOWLEDGEMENTS

I want to thank my supervisor Bengt Mannervik for making this possible. Tomas Nordlund for giving me all the attention I needed.Mats Persson for letting me use his laboratory when working with phages.Anders Undén for letting me work in his laboratory in my first attempts to make TS-analogs.Gun Stenberg for contributing to this work with very constructive suggestions and hersense of humor.Everyone helping me write this thesis.

I am very happy to have been a part of such a friendly group where everybody is socooperative. Thank you for all the fun times.

I specifically want to thank Maria & Fredrik and Ulrika & Micke for making Uppsala afriendly place.Peo for a lot of interesting discussions and of course for teaching me polska!Marianne and Ina who helped me get started on the project that led to this thesis.Ann for being very good at screening the literature and find me valuable information.Birgitta and Dan for keeping my mind on salsa and wine.Ylva for being such a dreadful RISK-player.Maryam for always standing by my side.



My old lab-group from Stockholm: Lizette, Camilla, Therese, Harvinder and Ingvar, whohave been a good support throughout the years.

All my sailing friends, I could not have done this without the self-confidence you haveencouraged.

Anki, Alexandra, Marcus and Lars for watching out for me.

I am grateful to my family Ernie, Kajsa, Anna and Maja for being so supportive.

I am very happy to have found Jens-Petter, the love of my life .

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