Bienzyme Complex Research Articles

Monovalent Metal Ions Play an Essential Role in Catalysis and Intersubunit Communication in the Tryptophan Synthase Bienzyme Complex

This investigation shows that the alpha 2 beta 2 tryptophan synthase bienzyme complex from Salmonella typhimurium is subject to monovalent metal ion activation. The effects of the monovalent metal ions Na+ and K+ were investigated using rapid scanning stopped-flow (RSSF), single-wavelength stopped-flow (SWSF), and steady-state techniques. RSSF measurements of individual steps in the reaction of L-serine and indole to give L-trytophan (the beta-reaction) as well as the reaction of 3-indole-D-glycerol 3'-phosphate (IGP) with L-serine (the alpha beta-reaction) demonstrate that monovalent metal ions such as Na+ and K+ change the distribution of intermediates in both the transient and steady states. Therefore the metal ion effect alters relative ground-state energies and the relative positions of ground- and transition-state energies. The RSSF spectra and SWSF time courses show that the turnover of indole is significantly reduced in the absence of either Na+ or K+. The alpha-aminoacrylate Schiff base species, E(A-A), is in a less active state in the absence of monovalent metal ions. Na+ decreases the steady-state rate of IGP cleavage (the alpha-reaction) to about 30% of the value obtained in the absence of metal ions. Steady-state investigations show that in the absence of monovalent metal ions the alpha- and alpha beta-reactions have the same activity. Na+ binding gives a 30-fold stimulation of the alpha-reaction when the beta-site is in the E(A-A) form.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric linkages between .beta.-site covalent transformations and .alpha.-site activation and deactivation in the tryptophan synthase bienzyme complex

This work examines two aspects of the catalytic mechanism and allosteric regulation of the tryptophan synthase bienzyme complex from Salmonella typhimurium: (a) the chemical mechanism by which indole and other nucleophiles react with the enzyme-bound alpha-aminoacrylate Schiff base intermediate, E(A-A), to form quinonoidal intermediates, E(Q), and (b) the effects of covalent transformations at the beta-site on the catalytic activity of the alpha-site. Transient kinetic studies in combination with alpha-secondary deuterium isotope effects are undertaken to determine the mechanism of nucleophile addition to E(A-A). These studies establish that nucleophilic attack is best described by a two-step reaction sequence consisting of a binding step that is followed by Michael addition to the conjugated double bond of E(A-A). Analysis of isotope effects suggests that the transition state for indole addition gives an E(A-A) beta-carbon that resembles an sp3 center, while the stronger nucleophiles, indoline and beta-mercaptoethanol, have transition states that appear to more closely resemble an sp2 beta-carbon. The effects of beta-site covalent transformations on alpha-site catalysis were studied using quasi-stable beta-site intermediates and the alpha-site substrate analogue 3-[6-nitroindole]-D-glycerol 3'-phosphate (6-nitro-IGP). It was found that the cleavage of 6-nitro-IGP is strongly activated by the formation of E(A-A) and various E(Q) species at the beta-site but not by external aldimine species. Therefore, we conclude that the conversion of the L-Ser external aldimine to E(A-A) is the beta-site process which activates the alpha-site, while conversion of E(Q) to the L-Trp external aldimine triggers deactivation of the alpha-site. These findings are discussed within the context of allosteric regulation of substrate channeling in tryptophan synthase catalysis.

Influence of .alpha.-Subunits on the High-Pressure Stability of Apo and Holo .beta.2-Subunits in the Bienzyme Complex Tryptophan Synthase from Escherichia coli

At a hydrostatic pressure of up to 2 kbar, the isolated alpha-subunit of tryptophan synthase from Escherichia coli proved to be a stable enzyme by virtue of specific activity as well as UV absorption and fluorescence emission spectra. The protein can therefore be regarded as a suitable effector for the investigation of structure-function relationships in the dimeric beta 2-subunit under the influence of high hydrostatic pressure. Complete deactivation of the beta 2-component in the alpha 2 beta 2 bienzyme complex occurs above 1300 bar (midpoint of transition for alpha apo beta 2, 790 bar; for alpha 2 holo beta 2, 1057 bar). Sucrose (13%) shifts both midpoints of transition to values higher by about 300 bar. As shown by sucrose gradient centrifugation and limited trypsinolysis, deactivation of the beta 2-dimer is paralleled by dissociation into denatured beta-chains. At 10 degrees C, the corresponding dissociation constants K at 1 bar as well as the reaction volumes of dissociation delta V are calculated as 4.2 x 10(-9) M and -196 mL/mol for the apo-beta 2-component and as 9.8 x 10(-19) M and -632 mL/mol for the holo-beta 2-component in the bienzyme complex. Furthermore, large negative activation volumes are determined, reflecting the rate increase with increasing pressure: -89 mL/mol for the apo-beta 2-dimer and -195 mL/mol for the holo-beta 2-dimer.(ABSTRACT TRUNCATED AT 250 WORDS)

Coordination of catalytic activities within enzyme complexes.

If two enzymes are physically and permanently associated as a bi-enzyme complex and if these enzymes catalyze non-consecutive chemical reactions, either of these reactions may inhibit or activate the other. If these reactions belong to two different metabolic cycles, the functioning of one of these cycles will control the fine tuning of the other. Thus simple kinetic considerations lead to the conclusion that, owing to the spatial organization of enzymes as multimolecular complexes, a fine tuning and a coordination of different metabolic networks, or cycles, may be exerted. It thus appears that channelling of reaction intermediates within a multienzyme complex does not represent the only functional advantage brought about by this type of spatial molecular organization.

A possible in vivo mechanism of intermediate transfer by glycolytic enzyme complexes: Steady state fluorescence anisotropy analysis of an enzyme complex formation

Rate constants of dissociation ( k off) and association ( k on) of the bienzyme complex yeast glyceraldehyde-3-phosphate dehydrogenase-yeast alcohol dehydrogenase have been determined in the absence and presence of NAD or NADH by fluorescence anisotropy measurements. We found that dissociation of the complex is considerably slower than catalytic turnover of either of the enzymes (that is k off ⪡ k cat) irrespective of the presence of coenzymes. A perusal of the literature reveals that this relation invariably applies to all systems studied so far. These observations all taken together constitute compelling evidence that direct metabolite transfer in enzyme complexes cannot be satisfactorily described by invoking the dynamic model but requires a model assuming more lasting complexes. This seems to support the case of the temporary-stationary model suggested by one of us. Implications of this conclusion are treated in depth and further evidence is cited under Discussion.

Evidence that mutations in a loop region of the alpha-subunit inhibit the transition from an open to a closed conformation in the tryptophan synthase bienzyme complex.

Rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy has been used to investigate the effects of single amino acid mutations in the alpha-subunit of the Salmonella typhimurium tryptophan synthase bienzyme complex on the reactivity at the beta-subunit active site located 25 to 30 A distant. The pyridoxal 5'-phosphate (PLP) cofactor provides a convenient spectroscopic probe to directly monitor catalytic events at the beta-active site. Single substitutions of Phe for Glu at position 49, Leu for Gly at position 51, or Tyr for Asp at position 60 in the alpha-subunit strongly alter the observed steady state and pre-steady state inhibitory effects of the alpha-subunit-specific ligand alpha-glycerophosphate (GP) on the PLP-dependent beta-reaction. However, similar GP-induced allosteric effects on the distribution of covalent intermediates bound at the beta-site that are observed with the wild-type enzyme (Houben, K.F., and Dunn, M.F. (1990) Biochemistry 29, 2421-2429) also are observed for each of the mutant bienzyme complexes. These results support the hypothesis that the preferred pathway of indole from solution into the beta-site is via the alpha-site and the interconnecting tunnel (Dunn, M.F., Aguilar, V., Brzović, P., Drewe, W.F., Houben, K.F., Leja, C.A., and Roy, M. (1990) Biochemistry 29, 8598-8607). Residues alpha E49, alpha G51, and alpha D60 are part of a highly conserved inserted sequence in the alpha/beta-barrel topology of the alpha-subunit. We propose that the GP-induced inhibition of the beta-reaction results, in part, from a ligand-dependent conformational change from an "open" to a "closed" structure of the alpha-subunit which involves this region of the alpha-subunit and serves to obstruct the direct access of indole into the tunnel. Our findings suggest that the altered kinetic behavior observed for the alpha-mutants in the presence of GP reflects an impaired ability of the modified bienzyme complex to undergo the conformational transition from the open to the closed form.

Allosteric interactions coordinate catalytic activity between successive metabolic enzymes in the tryptophan synthase bienzyme complex.

Tryptophan synthase from enteric bacteria is an alpha 2 beta 2 bienzyme complex that catalyzes the final two reactions in the biosynthesis of L-tryptophan (L-Trp) from 3-indole-D-glycerol 3'-phosphate (IGP) and L-serine (L-Ser). The bienzyme complex exhibits reciprocal ligand-mediated allosteric interactions between the heterologous subunits [Houben, K., & Dunn, M. F. (1990) Biochemistry 29, 2421-2429], but the relationship between allostery and catalysis had not been completely defined. We have utilized rapid-scanning stopped-flow (RSSF) UV-visible spectroscopy to study the relationship between allostery and catalysis in the alpha beta-reaction catalyzed by the bienzyme complex from Salmonella typhimurium. The pre-steady-state spectral changes that occur when L-Ser and IGP are mixed simultaneously with the alpha 2 beta 2 complex show that IGP binding to the alpha-site accelerates the formation of alpha-aminoacrylate [E(A-A)] from L-Ser at the beta-site. Through the use of L-Ser analogues, we show herein that the formation of the E(A-A) intermediate is the chemical signal which triggers the conformational transition that activates the alpha-subunit. beta-subunit ligands, such as L-Trp, that react to form covalent intermediates at the beta-site, but are incapable of E(A-A) formation, do not stimulate the activity of the alpha-subunit. Titration experiments show that the affinity of G3P and GP at the alpha-site is dependent upon the nature of the chemical intermediate present at the beta-active site.(ABSTRACT TRUNCATED AT 250 WORDS)

Reciprocal communication between the lyase and synthase active sites of the tryptophan synthase bienzyme complex

It is important to understand how the cleavage of indoleglycerol phosphate, which is catalyzed by the alpha subunits in the alpha 2 beta 2 bienzyme complex of tryptophan synthase, is modulated by the presence of L-serine in the beta subunits. Steady-state kinetic data, including the dependence of kcat on pH, allowed values to be assigned to each of the eight rate constants of the minimal catalytic mechanism. An ionizing group having an apparent pK value near 7.5 must be protonated for activity. The alpha active site ligands indolepropanol phosphate, glyceraldehyde 3-phosphate, and glycerol 3-phosphate increase both the affinity and the molar absorbance of L-serine and L-tryptophan bound to the beta active site. These effects prove that the alpha sites communicate with the beta sites over a distance of 30 A. 6-Nitroindole readily condenses with glyceraldehyde 3-phosphate, but not with L-serine. The turnover numbers for 6-nitroindoleglycerol phosphate and 6-nitroindole increased about 10-fold in both directions in the presence of L-serine bound to the beta 2 subunits. These data prove that the alpha and beta active sites communicate reciprocally and explain why the turnover number for the physiological reaction of indoleglycerol phosphate with L-serine greatly exceeds that of the cleavage reaction of indoleglycerol phosphate.

Mechanism of the physiological reaction catalyzed by tryptophan synthase from Escherichia coli

The physiological synthesis of L-tryptophan from indoleglycerol phosphate and L-serine catalyzed by the alpha 2 beta 2 bienzyme complex of tryptophan synthase requires spatial and dynamic cooperation between the two distant alpha and beta active sites. The carbanion of the adduct of L-tryptophan to pyridoxal phosphate accumulated during the steady state of the catalyzed reaction. Moreover, it was formed transiently and without a lag in single turnovers, and glyceraldehyde 3-phosphate was released only after formation of the carbanion. These and further data prove first that the affinity for indoleglycerol phosphate and its cleavage to indole in the alpha subunit are enhanced substantially by aminoacrylate bound to the beta subunit. This indirect activation explains why the turnover number of the physiological reaction is larger than that of the indoleglycerol phosphate cleavage reaction. Second, reprotonation of nascent tryptophan carbanion is rate limiting for overall tryptophan synthesis. Third, most of the indole generated in the active site of the alpha subunit is transferred directly to the active site of the beta subunit and only insignificant amounts pass through the solvent. Comparison of the single turnover rate constants with the known elementary rate constants of the partial reactions catalyzed by the alpha and beta active sites suggests that the cleavage reaction rather than the transfer of indole or its condensation with aminoacrylate is rate limiting for the formation of nascent tryptophan.

The tryptophan synthase bienzyme complex transfers indole between the .alpha.- and .beta.-sites via a 25-30 .ANG. long tunnel

The bacterial tryptophan synthase bienzyme complexes (with subunit composition alpha 2 beta 2) catalyze the last two steps in the biosynthesis of L-tryptophan. For L-tryptophan synthesis, indole, the common metabolite, must be transferred by some mechanism from the alpha-catalytic site to the beta-catalytic site. The X-ray structure of the Salmonella typhimurium tryptophan synthase shows the catalytic sites of each alpha-beta subunit pair are connected by a 25-30 A long tunnel [Hyde, C. C., Ahmed, S. A., Padlan, E. A., Miles, E. W., & Davies, D. R. (1988) J. Biol. Chem. 263, 17857-17871]. Since the S. typhimurium and Escherichia coli enzymes have nearly identical sequences, the E. coli enzyme must have a similar tunnel. Herein, rapid kinetic studies in combination with chemical probes that signal the bond formation step between indole (or nucleophilic indole analogues) and the alpha-aminoacrylate Schiff base intermediate, E(A-A), bound to the beta-site are used to investigate tunnel function in the E. coli enzyme. If the tunnel is the physical conduit for the transfer of indole from the alpha-site to the beta-site, then ligands that block the tunnel should also inhibit the rate at which indole and indole analogues from external solution react with E(A-A). We have found that when D,L-alpha-glycerol 3-phosphate (GP) is bound to the alpha-site, the rate of reaction of indole and nucleophilic indole analogues with E(A-A) is strongly inhibited. These compounds appear to gain access to the beta-site via the alpha-site and the tunnel, and this access is blocked by the binding of GP to the alpha-site. However, when small nucleophiles such as hydroxylamine, hydrazine, or N-methylhydroxylamine are substituted for indole, the rate of quinonoid formation is only slightly affected by the binding of GP. Furthermore, the reactions of L-serine and L-tryptophan with alpha 2 beta 2 show only small rate effects due to the binding of GP. From these experiments, we draw the following conclusions: (1) L-Serine and L-tryptophan gain access to the beta-site of alpha 2 beta 2 directly from solution. (2) The small effects of GP on the rates of the L-serine and L-tryptophan reactions are due to GP-mediated allosteric interactions between the alpha- and beta-sites.(ABSTRACT TRUNCATED AT 400 WORDS)

Kinetic misinterpretation of a coupled enzyme reaction can lead to the assumption of an enzyme—enzyme interaction

The time course of the conversion of 3-phospho-D-glycerate (GriP) to glyceraldehyde-3-phosphate (GraP) catalyzed by 3-phospho-D-glycerate kinase (GriP kinase) and glyceraldehyde-3-phosphate dehydrogenase (GraPDH) couple has been reinvestigated. The dependence of the steady-state rate on the dehydrogenase concentration is fully compatible with the consecutive nature of the reaction and therefore is not necessarily related to a complex formation of the two enzymes. To derive a Kd value of a bienzyme complex, as was done by Sukhodolets et al. [Sukhodolets, M. V., Muronetz, V. I. & Nagradova, N. K. (1987) Biochem. Int. 15, 373-379], is basically erroneous. In contrast with some previous reports, the maximal activity of GriP kinase is not influenced by the auxiliary enzyme present in the coupled assay system. Thus, no special accelerating effect can be attributed to GraPDH. 1,3-Bisphospho-D-glycerate (GriP2) bound to GriP kinase does not seem to be a substrate for GraPDH, providing evidence against channelling of GriP2 between the two enzymes.

Quantitation of the interaction between citrate synthase and malate dehydrogenase.

Formation of a bienzyme complex of pig heart mitochondrial malate dehydrogenase and citrate synthase in a buffered system is demonstrated by means of a covalently attached fluorescent probe to citrate synthase. Assuming 1:1 stoichiometry of the enzymes in the complex, an apparent dissociation constant of 10(-6) M was calculated from fluorescence anisotropy measurements. The effect of various metabolites on the interaction was tested. NAD+, oxalacetate, citrate, ATP, and L(-)- or D(+)-malate had no effect on the association of the two enzymes, whereas alpha-ketoglutarate increased and NADH decreased it. The interaction of mitochondrial citrate synthase with cytosolic malate dehydrogenase was found to be much weaker, whereas interaction of citrate synthase with another cytosolic enzyme, aldolase, could not be detected. In kinetic experiments, the activation of malate dehydrogenase by citrate synthase was observed. The effect of pyridine nucleotides and alpha-ketoglutarate is discussed in relation to the direction of the metabolic flow of oxalacetate.

Influence of cofactor pyridoxal 5'-phosphate on reversible high-pressure denaturation of isolated beta 2 dimer of tryptophan synthase bienzyme complex from Escherichia coli.

High hydrostatic pressure has been shown to cause reversible dissociation of the isolated apo beta 2 dimer of tryptophan synthase from Escherichia coli into enzymatically inactive monomers [Seifert, T., Bartholmes, P., & Jaenicke, R. (1982) Biophys. Chem. 15, 1-8]. Addition of the coenzyme pyridoxal 5'-phosphate affects the structural stability, as well as the kinetics of dissociation and deactivation. The apo beta 2 dimer is deactivated faster than the holoenzyme by a factor of 10. The midpoints of the corresponding equilibrium transition curves are observed at 690 and 870 bar, respectively. As shown by hybridization of native and chemically modified beta chains, the loss of enzymatic activity is accompanied by subunit dissociation. An additional deactivating effect is produced by the pressure-induced release of the cofactor from the holoenzyme. Renaturation after decompression has been monitored by circular dichroism and intrinsic fluorescence emission. Alterations of the dichroic absorption at 222 nm reflect the recovery of the native secondary structure, while tryptophan fluorescence represents a specific probe for the native tertiary structure in the immediate neighborhood of the active center of the enzyme. By application of both methods to monitor the reconstitution of the apo beta 2 dimer, two first-order processes may be separated along the time scale. The faster phase (k1 = 1.2 X 10(-2) s-1) yields a "structured monomer" with 85% native secondary structure and the tryptophan side chain buried in its native hydrophobic environment. As indicated by sodium borohydride reduction, this intermediate is able to interact with the coenzyme pyridoxal 5'-phosphate in the correct way; however, it does not show enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Phosphorus-31 nuclear magnetic resonance study of pyridoxal 5′-phosphate binding to [formula omitted] tryptophan synthase

The pyridoxal phosphate dependent bienzyme complex tryptophan synthase from Escherichia coli has been investigated using 31P nuclear magnetic resonance (NMR) at 72.86 MHz. In both the isolated β 2 subunit and the fully assembled α 2 holo β 2 complex, the pyridoxal phosphate 31P chemical shift is pH independent, indicating that the phosphate group of the cofactor is excluded from interaction with water and fixed in its dianionic form to the protein moiety. The line width of the phosphate signal of the coenzyme in the β 2 subunit is unusually large pointing to different equilibrium conformations of the protein. In the native holo complex, however, the corresponding line width may be interpreted to result from moderate mobility of the cofactor leading to a higher rate of the transaldimination step of the catalytic mechanism.

Equilibrium and kinetic measurements of the binding of pyridoxal 5'-phosphate to hybrid tryptophan synthase from Escherichia coli.

Hybrid tryptophan synthase was prepared that contains a β, subunit with one functional active site while the internal aldimine between cofactor and enzyme of the other protomer was reduced with sodium borohydride (α,ββ*). The modified enzyme shows a specific activity for the l‐serine+indole to l‐tryptophan reaction of about 50% compared to the native enzyme [Hathaway, G. M., Kida, S. and Crawford, I. P., Biochemistry 8, 989—997 (1969)]. The binding of pyridoxal 5′‐phosphate (pyridoxal‐P) to the α2apo ββ* complex and to the apo ββ* subunit was studied by equilibrium methods and rapid mixing experiments.The equilibrium binding curves for the hybrid species are hyperbolic (i. e. non‐cooperative), yielding apparent microscopic dissociation constants of 1.6±O.2 × 10−6 M for the α2 holoββ* subunit.The rate of formation of the internal aldimine in the unmodified active site was followed by fast reduction with sodium borohydride, L‐tryptophan synthesis was used to monitor the rate of formation of active enzyme.Cofactor binding to the α2apo ββ* complex is characterized by three sequential steps of decreasing rate: formation of a non‐covalent initial complex, which reacts to an enzymatically inactive internal aldimine, followed by a conformational change leading to the active α2holo ββ* bienzyme complex. A similar series of consecutive steps with decreasing reaction rates is observed for the binding of pyridoxal‐P to the apoββ* subunit. The formation of active enzyme, however, is biphasic. The faster process, which parallels the formation of the internal aldimine and which accounts for 85% of the total amplitude, obviously involves an already active high‐affinity state of the hybrid molecule whereas the second phase parallels the activation process as observed for cofactor binding to the native dimer.These findings are discussed with respect to the recently proposed mechanism for pyridoxal‐P binding to the unmodified apoβ2 subunit [Bartholmes, P. Balk, H. and Kirschner, K, Biochemistry 19, 4527–4533 (1980]

Interactions between Tryptophan Synthase from Escherichia coli and Derivatives of the Coenzyme Pyridoxal 5'-Phosphate

Abstract The interaction of the coenzyme analogues pyridoxal (A), pyridoxine 5'-phosphate (B), pyridoxic acid 5'-phosphate (Q and N-phosphopyridoxyl-L-serine (D) with both the isolated apo β2 subunit and the native < x2 apo β2 bienzyme complex of tryptophan synthase from Escherichia coli has been investigated using enzyme kinetics and CD spectroscopy. A 500-fold molar excess of (A) yields a maximum activation of the a2 apo β2 complex of 12% compared to the native holo bienzyme complex. The corresponding Michaelis constant KM equals 0.16 mM. Compounds (B -D) which lack the reactive carbonyl group in the 4-position cannot act as cofactor during enzymic turnover. However, they are competitive inhibitors with respect to the natural coenzyme pyridoxal 5'-phosphate. The corresponding inhibition constants K\ are for (B): 0.10 mM, (C): 0.03 mM and (D): 0.16 mM. The CD spectra of the aromatic side chains of both protein species ([@]278nm for the β2 subunit = 26 degr cm2 decimol-1, for the bienzyme complex = 40 degr cm2 decimol-1) remain un­ changed and no measurable dichroic absorption is induced in the visible region at 415 nm upon addition of (A), (at this wavelength productive binding of pyridoxal 5'-phosphate induces a significant extrinsic Cotton effect in the internal aldimine). Reaction with (B) leads to an enhancement of the dichroic amplitude at 278 nm of the isolated β2 subunit {A[6] = 6 degr cm2 decimol-1) and of the a2 β2 complex (zl[0] = 17 degr cm2 decimol-1) respectively. Com­ pound (C) shows no effect in the aromatic region of the β2 subunit, but a decrease in the a2 β2 complex (A[0) = 5 degr cm2 decimol-1). At 315 nm, however, a remarkable extrinsic Cotton effect of + 20 degr cm2 decimol-1 is induced in (Q . Ligand (D) causes a similar increase at 278 nm of A[0] = 14 degr cm2 decimol-1 in both protein species. The given data are discussed on the basis of the mode of binding of the natural coenzyme.

Circular dichroism studies on the interaction of tryptophan synthase with pyridoxal 5'-phosphate.

The interaction between the beta 2 subunit of tryptophan synthase and the coenzyme pyridoxal 5'-phosphate (PLP) is characterized by induced circular dichroism (CD) in the near-UV (260-285 nm) and in the visible region (320-480 nm, extrinsic Cotton effect). Because of its high mean residue ellipticity ([theta] = 56 deg cm2 dmol-1 for the isolated holo-beta 2 subunit and 102 deg cm2 dmol-1 for the alpha 2-holo-beta 2 complex, respectively) the latter has been used to define different conformational states of the beta 2 dimer via CD titrations. Fitting the obtained binding parameters to the known data from equilibrium dialysis leads to the result that the low-affinity state of the isolated beta 2 subunit shows a 3 times greater rotational strength than the holoenzyme in the high-affinity state. The generation of the final CD amplitude is characterized by a rate constant intermediate between the values for the formation of the internal aldimine and for the regain of enzymatic activity. Interaction of the alpha 2-apo-beta 2 bienzyme complex with the cofactor leads to a hyperbolic binding curve which is apparently free of contributions caused by unspecific PLP binding outside the active center. The determined dissociation constant of 9 x 10(-7) M is in good agreement with the value of 1 x 10(-6) M as obtained by equilibrium dialysis. Binding kinetics reveal a very slow process with a rate constant of 2.6 x 10(-4) s-1, significantly smaller than that for the regain of catalytic activity during reconstitution of the enzyme.

Identification of a small genetic region that encodes orotate phosphoribosyltransferase and orotidylate decarboxylase in Drosophila melanogaster

A variety of tests indicate that a small genetic region in chromosome 3R encodes at least a part of the bienzyme complex containing the last two enzymes of the pyrimidine biosynthetic pathway in Drosophila melanogaster, Complex U. Electrophoretic variants of the last pathway enzyme map to this region and both Complex U activities are proportionally reduced in flies heterozygous for specific deficiencies of the region. Complex U mutants have been isolated that lack both activities. Characteristics of these mutants are discussed relative to other pyrimidine pathway mutants known in Drosophila melanogaster and in mammals.