Bovine Kidney 2-Keto-4-Hydroxyglutarate Aldolase: Purification, Characterization, and Investigation of Catalytically Active Aminoacyl Residues.
Kitson, Richard Philip
1980
Abstract
The penultimate step in the mammalian catabolism of L-hydroxyproline to alanine and glyoxylate is catalyzed by the enzyme 2-keto-4-hydroxyglutarate (KHG) aldolase. This enzyme catalyzes the aldolytic cleavage of KHG to pyruvate and glyoxylate. KHG-aldolase has been isolated previously from extracts of bovine liver and E. coli. The enzyme from both of these sources has been purified to homogeneity and characterized extensively. However, low yields and problems with the reproducibility of the liver enzyme, led to the search for a better mammalian source of KHG-aldolase which could be used in comparative studies with E. coli KHG-aldolase. Such a source proved to be bovine kidney. An initial examination of the properties of bovine kidney KHG-aldolase indicated that it was similar to the liver enzyme in that it appeared to form a Schiff-base with substrate prior to catalysis: that is, mechanistically it is a Class I aldolase. In various other respects, (molecular weight, K(,m), turnover number, amino acid composition, and the ratio of aldolase to (beta)-decarboxylase activity), it appeared to differ from the enzyme as it was originally purified from liver. Recently, however, the liver enzyme has been purified in the presence of a protease inhibitor and found to have a molecular weight and amino acid composition equivalent to that of the kidney enzyme. The cysteinyl content and the availability of the cysteinyl sulfhydryl groups of the kidney enzyme were determined by the use of three different sulfhydryl reagents, p-chloromercuribenzoate (PCMB), 5,5'-dithiobis-(2-nitrobenzoate) (DTNB), and 4,4'-dithiodipyridine (DTDP). Three classes of groups were obtained: the first two classes of cysteinyl residues (6 residues out of a total of 24) were accessible to all three reagents, the remainder were accessible only to DTDP and PCMB in the absence of any denaturant. DTNB, even in the presence of 1.0% SDS, was only able to titrate a maximum of 12 cysteinyl residues. This information coupled with CD-spectra in the presence and absence of SDS indicate that these inaccessible residues might lie in an internal region of the protein which is resistant to distortion by SDS. In an attempt to uncover important functional residues at the active site, the activity of kidney KHG-aldolase was monitored as a function of the covalent modification of histidyl and cysteinyl residues by diethylpyrocarbonate and DTDP or PCMB, respectively. These aminoacyl residues were chosen because of prior indications with the original liver enzyme. The results were equivocal. There was a 50% loss of aldolase activity after the titration of 22 cysteinyl residues by PCMB, a 93% loss of activity after titration of 22 cysteinyl residues with DTDP, and no loss of aldolase activity after titration of 8 (out of 24 total) histidyl residues with diethylpyrocarbonate. In order to further clarify these results, kidney KHG-aldolase was inactivated with bromopyruvate, an active-site directed alkylating agent. The inactivation was found to be saturable with a K(,inact) = 18 mM and the maximal pseudo-first order rate of inactivation equal to 0.0176/sec. Pyruvate is a competitive inhibitor of the inactivation. Using {2-('14)C}bromopyruvate a maximum of 9 aminoacyl residues are modified, compared with a maximum of 2 when {2-('14)C}pyruvate is reductively bound to the enzyme. CD-spectra of the aldolase in the presence of pyruvate and bromopyruvate helped confirm that pyruvate and bromopyruvate interacted with the enzyme in a similar manner. Kidney KHG-aldolase, inactivated with {2-('14)C}bromopyruvate, was either oxidized with performic acid or reduced with sodium borohydride. After amino acid analysis of the modified enzyme, a radioactive peak corresponding to either S-carboxymethyl-cysteine or S-carboxyhydroxyethylcysteine was found.Types
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