1607-6729/03/0708- $25.00 © 2003 MAIK “Nauka / Interperiodica” 0225 Doklady Biochemistry and Biophysics, Vol. 391, 2003, pp. 225–228. Translated from Doklady Akademii Nauk, Vol. 391, No. 5, 2003, pp. 696–700. Original Russian Text Copyright © 2003 by Papisova, Bazhulina, Faleev, Demidkina. Pyridoxal-5'-phosphate (pyridoxal-P)-dependent enzymes catalyze various types of chemical transfor- mations of amino acids in pro- and eukaryotic cells, using the same cofactor. The specificity of substrate binding and the direction of the reactions catalyzed are determined by the properties of the protein components of the enzyme. To date, the spatial structures of many pyridoxal-P-dependent enzymes belonging to different classes have been determined. The analysis of the coen- zyme phosphate-group binding in three-dimensional structures belonging to five different types of polypep- tide chain folding showed that the majority of them contain a conserved serine or threonine residue whose hydroxyl groups form a hydrogen bond with one of the oxygen atoms of the phosphate group [1]. The contribu- tion of this serine (threonine) residue to catalysis has not been studied yet for any of the enzymes. Tyrosine phenol-lyase (EC 4.1.99.2) is a bacterial pyridoxal-P-dependent enzyme that catalyzes the reac- tion of L-tyrosine β-elimination to form phenol, pyru- vate, and ammonium [2]: Other natural and synthetic amino acids (such as L- serine, L-cysteine, S-alkyl-L-cysteines, β-chloro-L-ala- nine [2], and S-(o-nitrophenyl)-L-cysteine (SOPC) [3]) may be used as substrates in this reaction. NH 3 O – O HO + HO O O – O + + NH 4 . + In three-dimensional structures of the enzymes from Citrobacter freundii [4] and Erwinia herbicola [5], the hydroxyl group of Ser-254 is located at a distance of a hydrogen bond from one of the oxygen atoms of the coenzyme phosphate group. The purpose of this work was to study the contribu- tion of Ser-254 of tyrosine phenol-lyase to catalysis. We studied the spectral and kinetic characteristics of mutant forms of tyrosine phenol-lyase from C. freundii, obtained using site-directed mutagenesis, in which Ser- 254 was substituted with alanine (S254A enzyme) and cysteine (S254C enzyme). Binding of the coenzyme with mutant enzymes. The isolation and purification of mutant enzymes were performed as described for the wild-type enzyme [6]. Mutant apoenzymes were obtained by treating the mutant enzymes with D, L-penicillamine, as described in [7]. To clarify the role of substitutions in the coen- zyme binding by mutant enzymes, we determined the dissociation constant of the coenzyme. For this pur- pose, we added coenzyme aliquots to the apoenzyme solution and recorded the formation of the Schiff base (internal aldimine) between the aldehyde group of the coenzyme and the ε-amino group of the lysine residue in the active site. The formation of Schiff bases was monitored by the appearance of the absorbance with the maximum at 420 nm characteristic of aldimine. In each sample, the excess of aldimine was removed by ultrafiltra- tion on Centricon wells (P-30), and the amount of pyri- doxal-P in the enzyme solution and filtrate was deter- mined in 0.1 N NaOH, taking ∆ε 388 = 6600 M -1 cm –1 [8]. The difference in the content of pyridoxal-P in these two measurements corresponded to the amount of the protein-bound coenzyme. The results were processed using Scatchard plots [9]. The dissociation constant (ä d ) value for the mutant enzyme S254A was 1.5 × 10 –3 M. For the mutant enzyme S254C, this parameter could not be determined due to a lower affinity of the coenzyme for the apoenzyme, compared to the S254A enzyme. The ä d for the wild-type enzyme is 5.8 × 10 –7 M [10]. Therefore, the substitution of Ser-254 with Ala mark- edly decreases the affinity of the coenzyme for the apoenzyme. The decrease in free energy value as a result of the coenzyme binding with the mutant enzyme Tyrosine Phenol-Lyase: the Role of the Coenzyme-Binding Residue Ser-254 in Catalysis A. I. Papisova*, N. P. Bazhulina**, N. G. Faleev***, and T. V. Demidkina** Presented by Academician L.L. Kiselev March 18, 2003 Received March 21, 2003 * Department of Chemistry, Moscow State University, Vorob’evy gory, Moscow, 117234 Russia ** Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, ul. Vavilova 32, Moscow, 117984 Russia *** Nesmeyanov Institute of Organoelement Compounds, Russian Academy of Sciences, ul. Vavilova 28, Moscow, 117813 Russia BIOCHEMISTRY, BIOPHYSICS, AND MOLECULAR BIOLOGY