825. Transamination. Part II. The non-enzymic reactions between pyridoxamine and pyruvic acid and between pyridoxal and alanine

Abstract

The reaction between pyridoxamine and pyruvic acid in water a t 25" over the range pH 7-10 yields pyridoxal and alanine. Kinetic and spectroscopic studies show that the reaction proceeds by the fast and reversible formation of the Schiff's base. The rate-limiting step, which is general-acid catalysed, is the interconversion of this and the isomeric Schiff's base, i.e., the change *CH=N.CHMeCO,H *CH,*N=CMe*CO,H. The reverse reaction (formation of pyridoxamine and pyruvic acid) has also been studied, but in less detail. SNELL and his co-workers1 have shown that many of the enzymic reactions in which pyridoxal 5'-phosphate is involved as co-factor can be simulated in vitro by heating a mixture of an appropriate amino-acid, pyridoxal, and a metal-ion catalyst. Following earlier suggestions suggested that the transamination proceeds essentially as shown in scheme (A) (which for simplicity has been written without reference to ionic states or to the formation of chelates between the intermediates and any metal ions present in solution).The mechanism of the reactions is discussed. that imine intermediates are involved, Snell and his co-workers Intermediates of type (I) and their metal chelates (C,) have been i~o l a t e d .~ Semiquantitative kinetic studies 5 support the view that under Snell's conditions reaction proceeds via metal chelates CI and CLI. Metzler showed that in aqueous solution, imines * Banks, Diamantis, and Vernon:(e.g., I) are rapidly and reversibly formed from pyridoxal and amino-acids, and the equilibrium constants involved were measured spectrophotometrically. Matsuo found that similar reactions occur with pyridoxal5'-phosphate in ethanol and that, under these conditions, transamination occurs readily even in the absence of metal-ion catalysts. EXPERIMENTALMaterials.-Pyridoxamine hydrate crystallised from an aqueous solution of pyridoxamine dihydrochloride which had been neutralised with sodium hydroxide. It was washed with water and methanol and twice recrystallised, under nitrogen, from water (Found: C, 47.4; H, 8.1; H20, 16.6. Calc. for C8H,,N204: C, 47.0; H, 8.4; H20, 17.5%). The dissociation constants pKa,1, PKa,,, P K~,~, determined by titration with a glass electrode, were 3.33, 8-08, and 10.33, respectively.Pyridoxal ethyl acetal hydrochloride (Roche Products Ltd.) was dissolved in water and heated a t 60°, for 10 min.; pyridoxal separated after addition of an equivalent of sodium hydrogen carbonate and was recrystallised, under nitrogen, from water (Found: C, 57-2; H, 5.7. Calc. for C8H,N03: C, 57.5; H, 5.4%).DL-Alanine (B.D.H.), when recrystallised from aqueous ethanol, was chromatographically homogeneous.Commercial samples of N-methyldiethanolamine, NN-dimethylethanolamine, and triethanolamine were distilled under reduced pressure. Values of pK, were 8.61, 9-33, and 8.00 respectively.NN-Dimethylglycine hydrochloride was a commercial sample (Found : equiv., 140.4. Calc. for C4H,,C1N02: equiv., 139.6), pK, 9-70.Potassium pyruvate was obtained by adding freshly distilled pyruvic acid, b. p. ...

Some pyridoxal analogs and their transamination with amino acids

“…In addition, formation constants of ketimines of pyrldoxamine and pyruvate have been determined spectrophotometrlcally as a function of pH. These experimental values agreed with predicted formation constants calculated from pK'^ values of the dissociable ketimines and reactants (Banks _et , 1961).…”

“…A similar equation was derived for measuring apparent equilibrium constants for ketimine formation from pyridoxamine and pyruvate (Banks et al, 1961). Consider the above reaction where A is now keto acid, P = pyridoxamine, I = ketimine.…”

“…The pK'^ of 8.6 for the pyridinium group in pyridoxal changes to 5.9 In the imine, and the pK'^ of 4.2 for the phenolic group in pyridoxal changes to 10.5 in the imine, as a result of the formation of a rather stable hydrogen bond between the phenolic oxygen atom, the imine nitrogen atom, and perhaps the carboxylate group of the amino acid residue (Ac- Figure 4; Metzler, 1957;Christensen, 1958). In addition, the ionic equilibria of ketimines from pyridoxamine and keto acids have been determined spectrophotometrically ( Figure 5; Banks et al, 1961).…”

“…The existence of imines of pyridoxal and amino acids (and their metal chelates) has been proven by their isolation, identification, structure elucidation, and syntheses, which Figure 5. Absorption spectra and ionic equilibria of some ionic forms of ketimines of pyridoxamine and pyruvate (R = CH ) (Banks et al, 1961) 3…”

Catalysis of transamination by acetate

“…As the production ofextramitochondrial acetyl-CoA for fatty acid biosynthesis from either acetate or carbohydrate involves the action of acetyl-CoA synthetase (EC 6.2.1.1) or ATP citrate lyase (EC 4.1.3.8) respectively (Kornacker & Lowenstein, 1965), the activities of these enzymes have been measured in the liver, kidney cortex and perirenal adipose tissues ofthe lamb at three stages ofdevelopment: 140 days' gestational age and 1 day and 7 days after birth. Banks, Diamantis & Vernon (1961) found that the rate of transamination between pyridoxal and amino acids increased with buffer concentration; Bruice & Topping (1963a,b) showed that there was general acid-and base-catalysed transamination between amino and oxo acids. They suggested that the base removed the proton shown in the structure C=-N-C-H of the intermediate aldimine (cf. /\ Braunstein & Shemyakin, 1953;Metzler, Ikawa & Snell, 1954).…”