Enzyme ethanolamine ammonia lyase

The most extensive and informative enzyme work with B12 spin labels has been carried out on the enzyme ethanolamine ammonia-lyase 123). This work has employed six-coordinate 4-hydroxy-2,2,6,6-tetra-methylpiperidine-N-oxyl-5 -deoxyadenosylcobinamide. Ethanolamine ammonia lyase uses 5 -deoxyadenosylcobalamin as cofactor. Spin labeled 5 -deoxyadenosylcobinamide was used in order to determine the nature of the first step in the mechanism of action of ethanolamine ammonia lyase by determining the manner in which the Co—C bond is broken. Spin labeled 5 -deoxyadenosylcobinamide was synthesized by taking reduced diaquocobinamide and reacting it with an excess of 5 -tosyl-adenosine to give 5 -deoxyadenosylcobinamide. This was stirred for three days with a 20 fold excess of 4-hydroxy-2,2,6,6-tetramethylpiperidine... 

The application of magnetic resonance techniques to biological systems is a relatively new approach for the study of macromolecules. In this review we have presented the different approaches which have been made to study Bi2-enzymes. Clearly some progress has been made particularly from the application of ESR to a study of the enzymes ethanolamine ammonia-lyase and ribonucleotide reductase. Although 13C NMR is well in its developmental stages it is obvious that this technique will prove to be very useful for the examination of coenzyme-enzyme interactions. Studies of how corrinoids bind in enzymes and how sulfhydryl containing proteins are involved in enzyme catalysis comprise two major problems which must be overcome before realistic mechanisms can be presented for this group of enzymes. 

Deaminase. The adenosyl-cobamide-dependent enzyme ethanolamine ammonia lyase (from Clostridia) transforms the vicinal amino alcohol ethanolamine to acetaldehyde (with liberation of ammonia) (see Table 2, entry 3). Isotopic labeling has shown that the reaction is initiated by abstraction of a hydrogen atom from the hydroxymethylene group the reactive radical is provided from homolysis of the (Co-C)-bond of protein-bound coenzyme 2 (76). Magnetic field effects have provided independent evidence for the existence of caged radicals (85). 

Ethanolamine ammonia lyase, L-/ -lysine mutase, D-a-lysine mutase and ornithine mutase are representative of cobamide enzymes in which transfer of hydrogen occurs with cleavage of the C—N bond (Fig. 16). 

Ethanolamine ammonia lyase has a molecular weight of 520,000 and consists of 8 or 10 subunits. Two 5 -deoxyadenosylcobalamin molecular bind per enzyme molecule, and recent kinetic studies by Babior show that these two molecules carry out catalysis independently. Evidence is available that this enzyme functions by a radical mechanism since both spin labeling and Co(II) esr experiments indicate that Co(II) is an intermediate during H-transfer. Also, 5 -deoxyadenosine has been detected as a product of oxygenation of the enzyme-substrate complex (99—101). 

Spin labeled 5 -deoxyadenosylcobinamide has been used as a cofactor for ethanolamine-ammonia-lyase and the ESR spectrum followed during catalysis (123). This spin labeled coenzyme is biologically active in this enzyme. Enzyme kinetics showed this derivative to have the same Vmax as the cofactor 5 -deoxyadenosylcobinamide, but it has a higher Km value of 5.1 X 10-6 M compared to 4.6 X 10-6 for 5 -deoxyadenosylcobinamide (123). When the spin labeled coenzyme was incubated with apoenzyme to give the enzyme-coenzyme complex, the nitroxide ESR spectrum resembled that of free spin label but the lines are broadened significantly. 

Fig. 29. Decrease in intensity of nitroxide ESR signal npon addition of deuterated ethanolamine to ethanolamine ammonia lyase containing spin labeled cobinamide coenzyme. The two curves are for different concentrations of coenzyme to enzyme. The arrows indicate the point at which alcohol dehydrogenase and NADH was added to remove acetaldehyde from the enzyme. Note that full intensity is regained...

Tan, S. L., Kopczynski, M. G., Bachovchin, W. W., Orme-Johnson, W. H., and Babior, B. M., 1986, Electron spin-echo studies of the composition of the paramagnetic intermediate formed during the deamination of propanolamine by ethanolamine ammonia-lyase, and AdoCbl-dependent enzyme, J. Biol. Chem. 261 348303485. 

If ES involves a radical pair, the recombination rate of ES fe) is possible to be influenced by an external magnetic field. On the other hand, ki and k should be independent of the field. Harkins and Grisssom [4] studied MFEs on the conversion of unlabeled and deuterated ethanolamine to acetaldehyde and ammonia in bacteria by ethanolamine ammonia lyase. In this reaction, AdoCbP acts as a coenzyme and a radical pair is easily generated through the enzyme-induced homolysis of the C-Co bond. The escape 5 -deoxyadenosyl radical from the pair initiates the conversion reaction. They measured MFEs on the Vmax and Vmax/Km valucs at 25°C and obtained the results as shown in Fig. 15-4. The Vmax value was independent of B up to 0.25 T. This is reasonable because kj should be independent of B. On the other hand, the Vmax/ m values of the unlabeled and deuterated systems exhibited decreases of 25 % (at 0.1 T) and 60 % (at 0.15 T), respectively. These magnetically induced deceases can be explained by the HFCM, where k2 should be increased by such low fields as 0.1-0.15 T. At higer fields, the values were found to increase from their minimum... 

Diol dehydratase and ethanolamine ammonia lyase exhibit the largest overall tritium isotope effects that have been measured in Bi2-dependent enzymes [44, 45], the overall deuterium kinetic isotope effect is also substantial [10, 34, 45]. The observation of a deuterium isotope effect on the pre-steady-state formation of cob(ii)alamin in diol dehyratase [10] and in ethanolamine ammonia lyase [25] is consistent with kinetic coupling between the homolysis and H-transfer steps. 

Weisbiat, D. a., Babior, B. M. (1971) The mechanism of action of ethanolamine ammonia-lyase, a Bi2-dependent enzyme. Vlll. Further studies with compounds labeled with isotopes of hydrogen Identification and some properties of the rate-limiting step, J. Biol. Chem. 246, 6064-6061. 

Ethanolamine ammonia lyase, a coenz3me Bi2-requiring enzyme, catalyzes the following reaction ... 

Ethanolamine ammonia lyase (EAL) converts ethanolamine (2-hydroxy-ethylamine) to acetaldehyde, with the loss of ammonia. The cobamide bovmd to the enzyme of Clostridium sp. was identified as a pseudocoenzyme B12 (37) [221] but a range of other adenosyl-cobamides are accepted as cofactors. The active enzyme is multimeric, has an apparent molecular mass of about 560-600 kDa, but further studies are much less advanced with EAL than those of the AdoCbl-dependent enzymes described above. From ESR studies [222], and the fact that the cobalamin binding motif (Gly-X-X-His-X-Asp) is absent [223], EAL was concluded to be a base-on B -dependent... 

The small value of k 10" s" for the coenzyme is noteworthy in comparison to values of 2xl0 s" estimated for the rate-determining step in the diol dehydrase and ethanolamine ammonia lyase systems. This shows one of the problems in using the radical mechanism. This difficulty usually is explained by assuming that the enzyme somehow distorts the coenzyme so that the homolysis is about 15 kcal mol" more favorable in the holoenzyme. This distortion may destabilize the reactant and/or stabilize the transition state in order to hasten homolysis in the holoenzyme. For some time, it was thought that the axial base might promote these distortions, but Hay and Finke found that... 

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