Metal Ion Catalysis of the Transamination Reaction. a Thesis
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Metal Ion Catalysis of the Transamination Reaction. A thesis submitted to the University of London for the degree of Doctor of Philosophy, • by Trevor Matthews Chemistry Department, Bedford College, December 1$66. University of London, •» 1 ProQuest Number: 10098138 All rights reserved INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted. In the unlikely event that the author did not send a complete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest. ProQuest 10098138 Published by ProQuest LLC(2016). Copyright of the Dissertation is held by the Author. All rights reserved. This work is protected against unauthorized copying under Title 17, United States Code. Microform Edition © ProQuest LLC. ProQuest LLC 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106-1346 ' , Abstract Part I of the thesis deals with the formation and transamination of Schiff's base complexes of copper, pyridoxal phosphate and glutamate, and the transamination of reaction mixtures containing copper, pyridoxamine phosphate and a-ketoglutaric acid. The formation of the complex of copper,, pyridoxal phosphate and glutamate was found to be first order in both pyridoxal phosphate and glutamate and zero order in copper. Spectrophotometric studies showed isosbestiv points when reaction mixtures were scanned over the range 20,000-35f000 cm*"^ indicating that only one step is involved. At high concentrations of copper (^l6 mî-l) the initial reaction rate became slig h tly dependent upon the copper concentration, and the i n i t i a l optical density of the reaction mixture departed from that of pyridoxal phosphate. These deviations are explained by the postulation of an intermediate carbinolamine complex. ' The complex formed from copper, pyridoxal phosphate and glutamate transaminated to give pyridoxamine phosphate and a-ketoglutaric acid, • The presence of metal ions appears to catalyse the transamination reaction, copper being the most active. If account was taken of the fact that in the absence of metal ions the Schiff’s base of pyridoxal phosphate and glutanate is largely hydrolysed, however, the rate of transamination was found to be less in the presence of metal ions than in their absence. The transamination 6f reaction mixtures containing copper, pyridoxamine phosphate and a-ketoglutaric acid took place without significant formation of the Schiff’s base complex, due to the unfavourable 2a •" equilibrium constant for the formation of the Schiff's base in this system. Transamination was found to be very much more rapid than in the case of the copper complexes of the Schiff's base of pyridoxal phosphate and glutamate. The transamination of pyridoxamine phosphate and a-ketoglutaric acid is first"order in a-ketoglutaric acid (tailing off at higher concentrations of ..aKG) and exhibits a rate maximum at a copper concentration of tifice that of pyridoxamine phosphate. This maximum is justified mathematically by assuming th a t the concentration of a complex of copper and the Schiff's base from pyridoxamine phosphate and a-ketoglutarate + is very low, and that the complex accepts a further Cu ion2 at high concentrations of copper. The transamination of pyridoxamine phosphate was found to be ■ preceded by what at first appeared to be an induction period, further study of which indicated that the very small change in absorbance was caused by a rate limiting dehydration of the carbinolamine of pyridoxamine phosphate and cL-ketoglutarate. Low concentrations of carbinolamine; Schiff's base complex ; S c h iff's base and carbinolamine complex account fo r the f i r s t order nature of the reaction. The non-zero values of these concentrations probably cause the deviation from first order. Part II of the thesis is concerned ifith the evaluation.of the stability constants of the simple and mixed complexes of pyridoxal phosphate, pyridoxamine phosphate, glutamate and u-ketoglutarate. The pK values of pyridoxal.phosphate, pyridoxamine phosphate and CL-ketoglutaric acid (necessary for stability constant determinations) — 2b — were found by means of a potentlornetric titration method. The stability constants of copper and nickel with pyridoxal phosphate; copper, nickel, cobalt and zinc with pyridoxamine phosphate phosphate; and copper, nickel, cobalt and zinc with a-ketoglutaric acid were"also determined by a similar method. The stability constants as normally defined are shown to be meaningless where the compleit can accept protons at. p[ligand anion] valües of 0,5 and 1.5 and results obtained by the usual procedures have been converted to more meaningful re s u lts when the pK values of the complex are knovm. A graphical method of determining the stability constants of the complexes of the Schiff's base of pyridoxal phosphate and glutamate has been developed. The method, relies on absorbance readings taken at one wavelength o&ly. A graph is plotted of Log(Stability Constant) against assumed values of the extinction coefficient of the complexThe intersection of these lines for several sets of experimental conditions gives the required value of the stability constant. The equilibrium constant for the formation of the Schiff' s base of pyridoxal phosphate and glutamate, required in the above, was found by the usual graphical method at several pH values. A potentiometric titration ipethod was used to evaluate the stability constants of complexes of the type where P and G. represent pyridoxal (or pyridoxamine) phosphate and glutamate (or a-ketoglutarate) respectively, and where p and g can take values up to 2. A computer was used to solve the complex equations which were derived to describe the systen. - 2c - A cknovrl ed g em ent s The author wishes to thank the Medical Research Council, for the award of the Research Assistantship which made this work possible. He would also like to thank his supervisor, Dr. M.E.Farago, for her invaluable help and criticism during the production of this thesis. - 3 - Contents p 5 Introduction, 24 Reaction between pyridoxal phosphate, Ra glutamate and M(U), 42 Transamination of the complexes of pyridoxal phosphate and glutamate, 60 Reaction between pyridoxamine phosphate, a-ketoglutaric acid and M (ll). 82 Further reaction of pyridoxamine phosphate and a-ketoglutaric acid, 90 Stability constants of the complexes of pyridoxal phosphate, pyridoxamine phosphate and a-ketoglutarate vrith several metals. 97 Relationship between concentration and activity of the hydrogen ion. 102 The pK values of pyridoxal phosphate, pyridoxamine phosphate and a-ketoglutaric acid. ; 107 Results of stability•constant determinations. 123 The stability constants of the metal-Schiff's base complexes. 141 The equilibrium constant for Schiff Is base formation. 145 The mixed stability constants of Schiff's bases with several metals. 179 General discussion 181 Appendix I - Preparation of Schiff's base complexes. 187 Appendix I I - A bbreviations. 189 Appendix I I I - Buffers used in present work. 191 Appendix IV - Reagents used in the present yjork 192 Appendix V *• Program used to determine the mixed s ta b ility constants. - 4 - Introduction Transamination is one of a large group of reactions of amino acids (I) which are catalysed by pyridoxal phosphate (II) containing enzymes*(l,2). H T H I I I R—C-C~C~CO.,H 0= P— 0—CH I I I 2 X H RKo (I) (II) These include’ ( i) Elimination and replacement of substituents on the a-carboh, as in ' (a) Transamination (b) Dissociation of the a-hydrogen (c) Racémisation of a-amino acids (d) Oxydative deamination (e) Decarboxylation of a-amino acids (f) a,p clevage of p-hydroxyamino acids (g) Condensation of glycine (or serine) ^ An enzyme is defined as a protein with catalytic propertities due to its powers of specific activation, - 5 - and ( i i ) Elimination and replacement of substituents* on (3 and j'-carbon atoms. These apparently unrelated reactions have been rationalised by Metzler*s mechanism (p 12). Most of these enzymatic reactions have been duplicated in non-enzymatic 'model' systems (3-13)» in which pyridoxal^(usually non-phosphorylated) or other appropriate aldehyde (4,8,1?) and a suitable metal salt (6) serve as catalyst. Snell (4) has suggested that the catalytic potentialities of pyridoxal phosphate containing enzymes are those of their prosthetic groups*, and that enzymatic and non-enzymatic reactions proceed by closely similar mechanisms. Transamination is defined as the interchange of the functional groups =00 of an u-amino and an a-keto acid, as in equation (i). R-CH-NHg ^ . R'-CO ^ R-ÇO + R'-CH-NH^ ' CO^H ' CO^H ' CO^H COgH Biological transamination has been shown to require the presence of pyridoxal (14) or pyridoxal phosphate (15,16) which are knoim to + For abbreviations of these terms see Appendix II ^ The reactive groups attached to the enzyme, e.g. pyridoxal phosphate in transaminases. form Schiff's bases vrith a variety of amino acids. (18-22), It seemed probable, therefore, that transamination involved the steps:- f°2 R—CH— ;=GH R—CH—H=CH P OCH, .OH -H.0 P OCH_ 2 -CH, (III) ( i i ) CO R—C=0 m^CH^ R' i=K —CH 2 ■ P OCH;s..^::Ss_^H ' -H^O P OCH .OH -CH, (IV) and by a sim ilar mechanism f z R'—C=0 KH^— CHg Ri__CH— o=QH P OCH OH P OCH: .OH ( i i i ) •CH, CH, ^ 5 ' H ■ H Addition of equations (ii) and (iii) gives (i), the position of the • ■ ' . •I' equilibrium being decided by the thermodynamics of the individual - 7 - steps. (The is not shorn in the above equations but is believed to be attached to the phosphate group and the ring nitrogen (2) ). Hon-en7,yr'.r’cic transamination of pyridoxal by an a-amino acid was first shovn.