Biocatalyst Design for Stability and Specificity - ACS Publications

Chapter 14

Artificial Bifunctional Enzymes A Tool T o Improve Consecutive Enzyme Reactions and Cell Metabolism

Downloaded by UNIV OF PITTSBURGH on May 4, 2015 | http://pubs.acs.org Publication Date: December 11, 1993 | doi: 10.1021/bk-1993-0516.ch014

Leif Bülow Department of Pure and Applied Biochemistry, Chemical Center, P.O. Box 124, Lund S-221 00, Sweden

Two artificial bifunctional enzymes, β-galactosidase/galactokinase and β-galactosidase/galactose dehydrogenase, have been prepared by gene fusion and expressed in E. coli. The hybrid proteins are able to catalyze the hydolysis of lactose followed by either phosphorylation or oxidation of the galactose formed. Both hybrid enzymes exhibit favorable proximity effects when the coupled enzyme reactions are analyzed. The effect of the connecting segment on the function and stability of the bifunctional enzyme has been studied in vivo and in vitro. Artificial bifunctional enzymes are not only used in an isolated form but are also valuable tools in studies of cell metabolism to evaluate the aspects of proximity in metabolic maintenance and cell regulation.

SPATIAL ENZYME ORGANIZATION IN VIVO A living cell is a complex entity containing thousands of enzymes involved in the reactions necessary to maintain the cell status. Most biochemical reactions are organized into multistep pathways in which several enzymes can be involved (1). Physical arrangements among enzymes in such pathways are less pronounced in lower organisms but in eucaryotic cells many of the different metabolic pathways are frequently compartmentalized in organelles, for instance, the replication of D N A in the nucleus and the tricarboxylic acid cycle in the mitochondrion. Furthermore, it is believed that the enzymes of a specific reaction pathway within a single compartment are complexed in ordered or at least nonrandom arrangements (2). Highly organized enzyme systems in processive pathways such

0097-6156/93/0516-0174$06.00/0 © 1993 American Chemical Society

In Biocatalyst Design for Stability and Specificity; Himmel, M., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1993.


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Artificial Bijunctional Enzymes

as fatty acid synthesis and oxidation as well as nucleotide metabolism have been demonstrated. The enzyme interactions in these pathways are strong and thus have been easy to demonstrate. Such associations between enzymes are usually confirmed when the enzyme activities copurify and the ratio between the activities remains constant during the purification steps. Multienzymes capable of catalyzing several separate catalytic reactions have been characterized as either multienzyme complexes or multifunctional enzymes. A multifunctional protein is composed of a polypeptide chain(s) carrying two or more active sites. A multienzyme complex also has several active sites, however each on distinct polypeptide chains. These complexes often have a very low dissociation constant (3). Expressions like protein machines, enzyme clusters, supramolecular complexes, aggregates and metabolons are all referring to multienzymes. Recommendations for the nomenclature of these multienzymes have now been established and generally accepted (4,5). Multienzymes are often consecutive enzymes in a reaction path or part of a metabolic pathway, i.e. the product of the first enzyme will serve as a substrate for the second enzyme whose product will serve as the substrate for the subsequent enzyme and so forth: Substrate Enzyme 1»

χ Enzyme 2

>

2

Enzyme 3»

Most obviously, by aggregation of polypeptide chains into multienzyme complexes like the pyruvate dehydrogenase complex or multifunctional proteins like fatty acid synthase, the enzyme activities are gathered efficiently in a "microcompartment" . There is even evidence for specific interactions between many of the so called "soluble" consecutive enzymes in a pathway. Most attention has been focused on the two major metabolic pathways, the glycolytic pathway occurring in the cytosol and the tricarboxylic acid cycle in the mitochondria matrix. The complexation between the enzymes in glycolysis tends to be weak and thus difficult to demonstrate; the enzymes are associated but the organization is loose and only transient (6-8). For instance, direct transfer of NAD(H) between glycerol-3-phosphate dehydrogenase and lactic dehydrogenase has been shown in vitro (9, 10) but the interpretation of the data has been debated (77, 72). Other enzymes in glycolysis have also been investigated (73, 14). It has been claimed that the dynamic assembly and disassembly of the transiently existing complexes provide a regulatory mechanism for catalytic activity of the enzymes involved (75,16). It has further been suggested mat the association of glycolytic enzymes to the cytoskeletal proteins and actin containing structural elements of the cell is responsible for some compartmentalization in the cytoplasm (77, 78). The protein concentration in the mitochondrial matrix is over 50% and it has been proposed that the enzymes in the Krebs cycle probably also exist and behave as a multienzyme entity rather than as free enzymes in solution (79). Several publications have presented results indicating physical interactions between the enzymes in the cycle and metabolically related enzymes (summarized in (20)). It has also been possible to detect the existence of the Krebs cycle enzymes as a sequential complex (27). Moreover, organization of a reaction cycle is not restricted to only one compartment. The urea cycle has been shown to be structured in a sequential association that spans two cellular compartments, the cytoplasm and the mitochondria (22).


BIOCATALYST DESIGN FOR STABILITY AND SPECIFICITY

176 Importance of proximity


The arrangement of sequentially operating enzymes into multienzyme complexes and multifunctional enzymes involves several advantages for the reactions catalyzed. Different functional consequences of the organization of the enzymes into multienzyme systems have been proposed. Particularly features gained in cellular metabolism have been emphasized (2,20,23-26). Coordination effects. A n entire sequence of enzymes can be coordinately activated or inhibited. A n example of this phenomenon has been found for the arom conjugate, a multifunctional protein with five distinct consecutive enzymes residing on a dimer of a single polypeptide chain. Four out of the five enzyme activities are activated by the first substrate. In addition, all five activities appear to be coordinately protected from proteolysis when the first substrate is present (27). Compartmentation. A multienzyme system has the potential of compartmentalizing or containing the substrate of a pathway, which implies that the system prevents interference from an enzyme activity outside the metabolic sequence. This efficient transport of molecules from one enzyme to the next without complete equilibration with the surrounding fluid is also known as substrate channeling. Furthermore, labile intermediates can be protected. Elimination of lag phases. The intermediate substrate formed does not or only partially diffuses out into the surrounding medium, thereby a high local concentration of substrate for the second enzyme can be obtained. The transient time or the lag phase, defined as the time required to attain a steady-state rate in a series of reactions, is thereby also diminished. Reduction of diffusion times. The transient time, the time required for the product of one enzymatic reaction to diffuse to the active site of the next enzyme is decreased, particularly i f the surrounding medium is viscous (cf. the high protein concentration of the mitochondria). Biosynthesis is more efficient. The most effective coordination of the biosynthesis of enzyme molecules needed for a reaction path can be achieved if the genetic loci are combined into a single unit encoding a single polypeptide chain.

MODEL SYSTEM MIMICING MULTIENZYMES As previously indicated, the spatial organization of enzymes appears to be the rule rather than the exception in reflecting enzyme sequences in vivo. Therefore, it is important to further delineate the potential advantages gained by reconstruction of proximity between enzymes using different model systems. Three main different approaches have been utilized to mimic naturally occuring enzyme systems; coimmobilization of the enzymes to a solid matrix, chemical cross-linking of the enzymes in a random or an oriented fashion, and preparation of artificial multifunctional enzymes by gene fusion.

Co-immobilization Mosbach and Mattiasson (27) co-immobilized a two-enzyme system, hexokinase (HK) and glucose-6-phosphate dehydrogenase (G-6-PDH) which catalyze the sequential reactions:


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glucose

Artificial Bifunctional Enzymes

H K^^ glucose-6-phosphate ATP

G-6-PDH NADP

177

6-phospho-gluconolactone + NADPH


The initial rate of N A D P H formation was considerably enhanced when the two enzymes were co-immobilized, as compared with the situation when they were soluble or immobilized on separate beads (27). The two-enzyme system was extended to a three-enzyme system (28, 29) and later on the enzymes of the urea cycle were co-immobilized to agarose (30). The proximity of the enzymes made the immobilized systems more efficient than the soluble ones since the outdiffusion of the intermediates was hampered due to the unstirred layer surrounding the beads.

Cross-linking of enzymes Chemical cross-linking between sequentially operating malate dehydrogenase and citrate synthase in the citric acid cycle has been performed to analyze the kinetic behaviour of aggregated enzyme systems (31, 32). The kinetic advantages such as increased steady-state rate in the inital phase of the coupled reaction became apparent only when a crowding agent, polyethylene glycol, was included in the assay system. Improvements of the methodology by cross-linking were accomplished when the active sites of two dehydrogenases were spatially arranged face-to-face prior to cross-linking. Diffusion of the product of the first enzyme to the active site of the second enzyme was shown to be facilitated due to the proximity and proper orientation of the active sites (33).

Gene fusion Another efficient approach to obtain proximity between enzymes is to ligate the enzymes on the D N A level. The structural genes of the enzymes of interest are fused in-frame generating an artificial bifunctional enzyme carrying both active sites when the chimeric gene is expressed in a suitable host. Fusions can be made either to the amino- or carboxy-terminal regions of the proteins depending largely on the availability of suitable restriction enzyme sites on the corresponding structural genes. If no such restriction sites are accessible at the 5'- or the 3'- ends of the genes they can be generated by site-directed mutagenesis. B y using chemically synthesized D N A fragments in the cloning procedure, special properties in the linker region between the enzymes can be designed by the selection of a certain oligonucleotide sequence. In the construction of artificial fusion enzymes, the three-dimensional structure of the enzymes is most often unknown, but frequently the C- and N-terminus are surface located. A gene fusion therefore normally does not or only to a minor extent interfere with the folding of the protein. If subunit interactions are disturbed or disrupted the fusion can often simply be made at the other end of the gene. The construction prepared is then inserted into a proper expression vector and transformed into a suitable host cell. The effects caused by the bifunctional enzymes on enzyme catalysis can then be analyzed either in vivo or in vitro. in many instances, the use of this genetic approach is advantageous over the immobilized and cross-linked enzyme systems. Large amounts of homogeneous bifunctional protein can be produced whereas the degree of crosslinking and homogeneity may vary between different preparations of chemically prepared enzyme conjugates. Additionally, much of the enzyme activity is often lost in the immobilization or cross-linking procedure which is normally not the case for gene fusion. Most often at least 50 % of the wild-type enzyme activity is retained if the entire primary structure of the native enzyme is maintained in the hybrid enzyme


178


prepared 2-5 amino acid residues can frequendy be removed from the terminus of the enzymes without affecting their activity. However, the ratio between the enzyme activities is more or less fixed in a genetically prepared system while it is easy to change it using the other two methods.

ARTIFICIAL BIFUNCTIONAL ENZYMES


Over the years we and others have prepared a number of different artificial bi- and polyfunction^ enzymes (for review see (34,35)). In this paper I will focus on two constructions, β-galactosidase/galactokinase and β-galactosidase/galactose dehydrogenase, which both illustrate several important properties associated with such hybrid enzymes.

β-galactosidase/galactokinase In the first attempt to construct an artificial bifunctional enzyme in vitro a fusion was made between β-galactosidase and galactokinase of E. coli, a tetrameric and monomelic enzyme, respectively (36). The 5'-end of galKv/as fused to the 3'-end of the structural gene of β-galactosidase, lacZ. The bifunctional gene product carried both activities although the β-galactosidase activity was reduced substantially due to 14 missing amino acids in the C-terminus. By adding those residues in a later construction the β-galactosidase activity could be restored (37). The bifunctional enzyme β-galactosidase/galactokinase had a tetrameric configuration (Figure 1) and could efficiendy catalyze the reaction sequence: lactose —galactose + glucose

^22?—^ galactose-1 -phosphate + A D P

This system was initially used as a model in studies of proximity effects between consecutive enzymes in vitro. By utilizing a third enzyme, galactose dehydrogenase, which is competitive to galactokinase, it was demonstrated that the galactose formed is channeled between the enzymes. The vicinity effect became more pronounced relative to a control of native enzymes when polyethylene glycol was used as a crowding agent in the reaction medium.

The linker region In naturally occuring multifunctional enzymes no homology between the linker regions has been observed (38) and the importance of these regions is still unclear. It has been suggested that the correct folding of the protein domains in the polyfunctional arom enzyme (S. cerevisiae) depends on the linker due to pauses induced by rare combinations of codons near the domain boundaries (39). Suitable oligopeptides as candidates for general gene fusion have been presented after the examination of pronounced characteristics of linker peptides joining domains in known tertiary protein structures (40). It has been reasoned that the embodied amino acid residues should give the linker some flexibility and allow it to interact with the solvent. In order to investigate the role of the linker to the function of an artificial bifunctional enzyme, further constructions of β-galactosidase/galactokinase fusion enzymes were made starting from the previous construction (37). Oligopeptides of different length and character were introduced between the enzymes. The choice of linker proved to be very important for both the expression of the fusion protein and its stability. Fusion proteins with polyglycine and polyproline linkers encoding polyproline and polyglycine, respectively, were not expressed to the same extent as fusion proteins with amino acids more randomly chosen.



Figure 1. Schematical configuration of the p-galactosidase/galactokinase complex.


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Oligonucleotide stretches of the same bases such as oligodC or oligodG were found to be difficult to express in an E. coli host Proteolytic degradation took place both intracellularly as well as extracellularly during purification. This proteolysis occured mainly in the linker region between the catalytic parts. The same phenomenon has been reported for other β-galactosidase fusion proteins as well, e.g. protein Α/β-galactosidase (41). Linkers with multiple glycine residues in the β-galactosidase/galactokinase hybrids were shown to be the most susceptible ones. Sequences such as gly-gly-X have been shown to be proteolytic processing sites in some biological systems (42). Furthermore, glycine is believed to be a flexible residue and thus might destabilize the linker. Several independent strategies to stabilize proteolytically sensitive fusion proteins have been presented (43).

Artificial bifunctional enzymes in vivo From all the reports dealing with enzyme interactions in vitro a lot of information about cellular organization has been derived. However, as the knowledge of cell metabolism becomes more detailed, the inherent complexity of living systems also becomes more apparent To understand the importance of a certain enzyme in a metabolic pathway, further studies in vivo will no doubt be required. When enzymes are removed from the cells several differences arise that can change the enzyme reactions (26): -

-

There is a potential loss of organization and compartmentation. Enzymes are diluted which can affect a number of interactions between macromolecules and small molecules and ions. The relative concentration of enzymes and substrates will change. In vivo the enzymes are often present in concentration comparable with or greater than their substrates. The kinetic parameters, K and y , could be significantly altered in vivo (13). m

max

Furthermore, the environment of the in vitro experiments is quite different from the in vivo conditions. For instance, the protein concentration is often much higher. It has been estimated to be 200-400 mg/ml (44). This high intracellular protein concentration promotes protein-protein interactions that would not occur in diluted solutions. This volume exclusion effect has been mimicked by the use of polyethylene glycol in vitro as previously described. Therefore, in vitro studies have to be extended to include studies on in vivo systems. Cell permeabUization is a widely used procedure where it is generally assumed that the protein organization is intact. A simple approach that allows noninvasive studies of cellular metabolism is N M R (45,46). -NMR studies of yeast cells have provided evidence that substrate channeling between the tricarboxylic acid cycle enzymes does occur supporting previous in vitro results (47). The different β-galactosidase/galactokinase fusion proteins were used as model systems to evaluate the importance of proximity between two metabolically related enzymes, β-galactosidase and galactokinase catalyze the first steps of lactose metabolism in E. coli. To study the function of the different hybrid enzymes in vivo, E. coli harbouring various β-galactosidase/galactokinase fusion enzymes were grown on minimal media with lactose as carbon source. Almost no differences in growth rate were visible. However, on introduction of a second plasmid encoding galactose dehydrogenase that scavenges on galactose, pronounced differences became apparent. The product of galactose


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181

dehydrogenase, galactono-lactone, is a metabolic end-product Therefore, faster growth rates were expected for E. coli cells in which the intermediate galactose can be efficiendy transferred or channeled to galactokinase. It was demonstrated that E. coli cells carrying p-galactosidase/ galactokinase fusion enzymes with short linkers (10 amino acid residues) spacing the two catalytic parts displayed faster growth rates as compared to the cells with bifunctional enzymes with longer linkers (20 residues) or two separate wild-type enzymes. The same behavior due to proximity was also observed by in vitro studies on the purified enzymes and crude extracts from cells producing the different p-galactosidase/galactokinase fusion proteins (48).


β-galactosidase/galactose dehydrogenase In order to further characterize artificial bifunctional enzymes a fusion between two oligomeric enzymes was carried out In this case there is a potential risk of protein polymer formation when the assembly of subunits takes place. However, the fusion of tetrameric β-galactosidase (E. coli) to the C-terminus of dimeric galactose dehydrogenase (Psedomonas fluorescens) resulted in completely soluble protein complexes (49). The oligomeric structure followed that of native βgalactosidase and consisted mainly of tetramers and hexamers. The hinge region, spacing the two enzymes, was only three amino acid residues and it is possible that a longer linker would change the quaternary structure.The galactose dehydrogenase moiety of the complex proved to be more thermostable than its native counterpart while the opposite was true for β-galactosidase. The potential proximity effects caused by the fusion were investigated by analyzing the kinetics of the coupled enzyme reaction using purified enzymes. The fused enzyme system carried out this reaction more efficiendy than a corresponding system composed of native enzymes with the same activities. The transient time of the coupled reaction was hence markedly reduced with the bifunctional enzyme. Furthermore, the overall reaction rate turned out to be higher in the fused system compared with the native system. The reason for the latter effect is not fully understood. A plausible explanation can of course be that substrate channeling is taking place between the active sites. The efficiency of the channeling was found to be dependent on the ratio of the activities between the two enzymes. When the activities of galactose dehydrogenase to β-galactosidase was equal only very small differences in catalytic behavior in comparison with the wild-type enzymes were observed. However, i f the ratio was increased (8:1) the difference became pronounced resulting in a two-fold increase in the steady-state rate of the coupled reaction for the fused system compared to the native. This shift in channeling could be achieved simply by changing the p H of the reaction buffer since galactose dehydrogenase has an alkaline p H optimum while β-galactosidase has its optimum at neutral pH. In this context the kinetic parameters KJJJ and v must also be considered. A shift in K for both lactose and galactose was observed. However, these changes can contribute only partly to the observed differences in kinetic behavior between the fused and native enzyme systems. Similar changes in K upon complexation have been observed in natural systems m a x

M

M

as well. For instance, a decrease in K values has been observed when the

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