Ala. AT was purified from pig heart by a new procedure , which consisted of an homogenisation, ammonium sulphate fractionation, heat treatment and a second AS fractionation, followed by DEAE cellulose and Sephadex G200 chromatography Thee purified enzyme had a specific activity of 450 units/mg protein, and appeared to be at least 95 % pure, from electrophoresis. Sodium dodecyl sulphate - polyacrylamidee gel electrophoresis indicated a submit molecular weight of 52000. The concentration of' the coenzyme was measured: it was 1 mole/54 00 gm protein. The kinetic parameters for four different assays and the inhibitor constants for several inhibitors were determined, at 25°C, at several pHs. These parameters were found to vary considerably with buffer concentration. For this reason, the values of these parameterswere extrapolated to zero buffer concentration. From a study of the affinities of several different assays and the inhibitor from ala AT, at pH 8.0, it seems that the ˂(carboxyl end side group of the substrate, (as well as the <.amiro and ˂ (keto groups), are involved in binding to the enzyme. A Dixon analysis of the results shows the existence of certain catalytically important pKs in the free forms of the enzyme, the enzyme-substrate complexes, and the enzyme-inhibitor complexes. The nature and role of the enzyme groups that are involved in.substrate binding are deduced, in part. Visible absorption spectra were recorded at 25°C. PL-ala AT shows a spectral pK at pH 7.6. This pK is raised to about pH 8 by fumarate or acetate binding. The dissociation constants obtained are similar to the inhibition constants. TSC binding produces an intense absorbance peak at 390 nm: a similar result has been obtained with asp AT. Substrates were titrated with ala AT and spectra of the enzyme-substrate complexes were calculated. An absorbance peak at 490 nm is observed. It is very probably a deprotonated, (highly conjugated), complex: i.e. there are separate deprotonation and protonation steps. A rough estimate is made for the relative concentrations of the different spectral forms of the enzyme-substrate complex, at pH 7.0. The pH dependence of the L alanine-pyruvate equilibrium enzyme-substrate complex spectrum was studied. The changes between pH 5.5 and. 10.5 can be ascribed to three pKs, and were used to explain the pKs of the complex, that were shown by kinetic analysis. The inactivation of the enzyme was studies using heat, (65°C), 5.6 M urea, formaldehyde, nitrite, TNM, and. photo-oxidation by rose Bengal and methylene blue. Both methylene blue photo-oxidation and TNM inactivation of ala AT give results similar to those obtained by 1Martinez-Carrion, for asp AT. With methylene blue, an essential residue is modified, which has a low pK end is believed to be a catalytic base; it is probably a histidine. With rose Bengal phot-oxidation, nitrite and TNM, inactivation is incomplete and the nature end role of the modified residue is v most uncertain. The results with formaldehyde inactivation suggest that it inactivates by a reaction at the active site: the possibility that this reagent cross-links a histidine end a lysine group is discussed. The inactivation results, all together, indicate that two different conformational changes can occur, when substrate is bound to ala AT. The nature of these changes and their relation to the kinetic results are discussed. A detailed model of the catalytic activity of ala AT is presented.
| Identifer | oai:union.ndltd.org:bl.uk/oai:ethos.bl.uk:595026 |
| Date | January 1973 |
| Creators | Austin, Ian Maxwell |
| Publisher | University of Warwick |
| Source Sets | Ethos UK |
| Detected Language | English |
| Type | Electronic Thesis or Dissertation |
| Source | http://wrap.warwick.ac.uk/72005/ |
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