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Phosphate and Analogues of the Substrate-Coenzyme Complex to Tyrosine Decarboxylase

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Phosphate and Analogues of the Substrate-Coenzyme Complex to Tyrosine Decarboxylase Biochem. J. (1980) 185, 41-46 41 Printed in Great Britain The Binding of the Coenzyme Pyridoxal 5'-Phosphate and Analogues of the Substrate-Coenzyme Complex to Tyrosine Decarboxylase Aldo ORLACCHIO,*$ Carla BORRI-VOLTATTORNI* and Carlo TURANOt *Istituto di Chimica Biologica, Facoltai di Farmacia, Universita di Perugia, Via del Liceo, 06100 Perugia, Italy, and tIstituto di Chimica Biologica, Facolta di Farmacia, Universita di Roma, 00100 Roma, Italy (Received 10 May 1979) Phosphopyridoxyl derivatives, which are stable analogues of a substrate-coenzyme complex, are bound at the active site with great affinity. From a comparison of the inter- action ofa number ofsuch compounds with the apoenzyme the AGO values for the binding of the substrate carboxy and phenyl groups and of the coenzyme aldehydic group were determined to be equal to (or more negative than) -3.8,-8.4 and -12.5 kJ/mol (-0.9,-1.9 and -3 kcal/mol) respectively; the AGO for the binding of the coenzyme phosphate group was shown to be more negative than -20.5kJ/mol (-4.9 kcal/mol). Two features of the binding process of the coenzyme-substrate analogues to tyrosine decarboxylase have already been found in the case of tyrosine aminotransferase [Borri-Voltattorni, Orlacchio, Giartosio, Conti & Turano (1975) Eur. J. Biochem. 53, 151-160]: (1) in the binding of the substrate to the enzyme a significant fraction of the intrinsic AGO appears to be used for some associated endoergonic process; (2) the AH0 and AS0 of binding appear to be very sensitive indicators of the correct alignment of the substrate-coenzyme analogues at the active site. Phosphopyridoxyl-amino acids are compounds Materials and Methods structurally similar to one ofthe proposed coenzyme- Tyrosine decarboxylase was extracted from cells substrate complexes that are formed at the active of Streptococcus faecalis obtained from Sigma site of pyridoxal 5'-phosphate-dependent enzymes. Chemical Co. (St. Louis, MO, U.S.A.); the cells In a previous paper (Borri-Voltattorni et al., 1975) we were grown in a vitamin B-6-deficient medium, so described the interaction of these compounds and of that the enzyme was mostly in the apoenzyme form. the coenzyme with apo-(tyrosine transaminase) from To remove traces of pyridoxal 5'-phosphate from rat liver, showing that they bind at the active site and the cells, the procedure of Maruyama & Coursin have a very high affinity for the enzyme; it was also (1968) was used; the only modification was to use possible to measure the contribution to the binding 10mM-sodium acetate buffer, pH 5.5, containing from different functional groups of the coenzyme 5mM-EDTA but without mercaptoethanol and and substrate. It was noticed that the AH0 and AS0 tyrosine for the last washings. The cells were then values of binding are particularly sensitive to the suspended in SmM-sodium acetate buffer, pH5.5; formation of a correct coenzyme-substrate-enzyme carborundum powder was added, and the suspension complex, and much more so than the AG0 values of was treated in a tissue disintegrator (Mickle Labora- binding. It seemed worthwhile to study the interaction tory Engineering Co., Gomshall, Surrey, U.K.) for of the same analogues with another vitamin B-6- h at maximum speed. The suspension was then dependent enzyme with the same substrate specificity, centrifuged at 140000g (ray. 5.9 cm) for 10min at 4°C but a different catalytic function. In the present in a Beckman Spinco ultracentrifuge; the apoenzyme paper we have therefore studied the binding of these was present in the clear supernatant, which was used compounds (Fig. 1) to tyrosine decarboxylase (L- as such. The activity remained unaltered for at least tyrosine carboxy-lyase) from Streptococcus faecalis a month if the solution was kept at 2-40C and for at (EC 4.1.1.25). least 3 months if kept at -20°C. The activity was measured by following the method described by Salvadori & Fasella (1970) for t To whom correspondence and requests for reprints glutamate decarboxylase, which uses a pH-stat for should be addressed. measuring the amount of CO2 liberated. Vol. 185 0306-3275/80/010041-06 $1.50/1 42 A. ORLACCHIO, C. BORRI-VOLTATTORNI AND C. TURANO N+H2-R R ICompound CH2 -CH3 (I) 0- CH3 O -O CH2-0-P -CH (II) GO2- H3C NZ H+ -CH2-CH / OH (III) -CH-CH2 /-OH (IV) CH20H -CH-CH2/ (V) I - CO2- -CH-CH2 I (VI) H -CH-CH2 OH (VII) I GOL2-- /0 - (VIII) p==O 0O- Fig. 1. Structures ofsubstrate-coenzyme complexes See Table 1 for names of compounds. The reaction mixture contained 250,umol of NaCI, needed to keep the pH constant. The dissociation l0pmol of L-tyrosine, pyridoxal 5'-phosphate in the constant of the pyridoxal 5'-phosphate and the range 0.075-0.250nmol, the inhibitor (when needed) inhibition constant K, for pyridoxal derivatives and and the enzyme in a volume of 5 ml. The concen- coenzyme analogues were determined as described in trations of substrate, coenzyme and inhibitor were a previous paper (Orlacchio & Borri-Voltattorni, close to the respective Km or K, values. 1979). All solutions were adjusted to pH 5.5 before use. A rate equation for tyrosine decarboxylase has All components, except the enzyme, were preincub- also been proposed, which considered the presence ated at the desired temperature in the vessel of the of a competitive inhibitor towards pyridoxal 5'- pH-stat (Radiometer TTT2) for 7min; the reaction phosphate (Orlacchio & Borri-Voltattorni, 1979). was then started with the addition of the enzyme This equation was used in the present work in order solution. The reaction was followed (for at least to measure the K, values of the pyridoxal derivatives; 10-15min) by monitoring the addition of 5mM-HCI K1 values were calculated by determining the recip- 1980 ANALOGUES OF SUBSTRATE-PYRIDOXAL PHOSPHATE COMPLEXES 43 rocal of the initial rate as a function of the reciprocal 7.0 1 8.0 of the coenzyme concentration, in the absence and in the presence of the inhibitor. The ratio of the slopes obtained in the presence and the absence of the inhibitor is equal to I +([I]/K,), from which K, can easily be calculated (Orlacchio & Borri- Voltattorni, 1979). No difference in inhibiton was found when the 7.5 apoenzyme was preincubated with the inhibitor; thus the activities of the apoenzyme treated with 1.25 pM- 6.5 I 5'-phosphopyridoxyl-L-tyrosine without preincub- 02 ation or with 5 min or 15 min preincubation differed by less than 3 %. 0.0 x Pyridoxal 5'-phosphate and pyridoxamine 5'- 4. phosphate were commercial products from Merck 7.02 A.G. (Darmstadt, Germany). The synthesis and the structure of the phosphopyridoxyl derivatives have 6.0 I 0.v OA been described in a previous paper (Borri-Voltattorni 10S8e et al., 1975). All other reagents were of analytical k grade. .2 6.5 bo Results 60 O All pyridoxal derivatives tested behave as com- 5.5 - petitive inhibitors towards the coenzyme. The AGO values of binding of the inhibitors were calculated from the K, values at 37°C, and are shown in Table 1. Effect oftemperature on theformation ofthe complexes 16.0 The variation of K, as a function of temperature 5.0 F between 10 and 37°C was also examined. Van't Hoff Af"' plots ofthe results obtained in this temperature range a---- are linear, within experimental error, indicating that the heat capacity change, AC, is very low. From these plots the AHO values for the binding of the inhibitor were calculated (Fig. 2). The estimated error for the AGO values is +5% and that for AH0 3.3 3.4 ±15%. The values of AGO, AHO and ASO (calculated 3.2 from AGO and AHO) of binding are shown in Table 1. 103/TI(K-') Fig. 2. Van't Hoffplots of 5'-phosphopyridoxyl derivatives m, Pyridoxal 5'-phosphate-dopa compound; *, 5'- Discussion phosphopyridoxyl-methylamine; 0, 5'-phosphopyrid- In interpreting the kinetics of tyrosine decarboxyl- OXyl-L-phenylalanine; l, 5'-phosphopyridoxyl-L- alanine; A, 5'-phosphopyridoxyl-L-tryptophan; v, ase, the non-enzymic formation of the Schiff base 5'-phosphopyridoxyl-tyramine; v, 5'-phosphopyrid- between tyrosine and pyridoxal 5'-phosphate must oxyl-L-tyrosinol. The values of logK, for 5'-phos- be taken into account (Orlacchio & Borri-Voltattorni, phopyridoxyl-L-tyrosine (o) refer to the right-hand 1979). A rate equation was therefore proposed, which scale, all the others to the left-hand scale. fitted well the experimental data. This rate equation is also compatible with a direct binding of the non- enzymically formed Schiff base with the enzyme active site. However, it must be pointed out that the occurrence of this binding is not yet proved; the same postulated substrate-coenzyme intermediate com- conclusion was reached by Litwack & Cleland (1968), plex, and have been shown to have a high affinity for when a similar binding of the Schiff bases was the apoenzyme. Being stable compounds, they are proposed for tyrosine aminotransferase. not transformed when bound at the enzymic active The pyridoxal derivatives that are the subject of centre and the equilibrium constant of their binding the present paper are structurally similar to the can be measured with precision. Vol. 185 A. ORLACCHIO, C. BORRI-VOLTATTORNI AND C. TURANO Table 1. Apparent thermodynanic parameters ofthe binding ofcoenzyme and coenzyme derivatives to tyrosine decarboxylase Details are given in the text. AGO AH0 ASO [kJ/mol [kJ/mol [J/mol per degree Coenzyme and derivatives (kcal/mol)] (kcal/mol)] (cal/mol per degree)] 5'-Phosphopyridoxyl-methylamine (I) -29.3 (-7.0) -50.2 (-12.0) -67 (-16) 5'-Phosphopyridoxyl-L-alanine (II) -33.1 (-7.9) -36.0 (-8.6) -8 (-2) 5'-Phosphopyridoxyl-tyramine
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