title: Precursor of beta-lactamase is enzymatically inactive. Accumulation of the preprotein in Saccharomyces cerevisiae.
abstract: Synthesis and properties of the bacterial precursor of beta-lactamase (E.C.3.5.2.6) were studied in Saccharomyces cerevisiae transformants. A protease-deficient yeast mutant was transformed with the plasmid pADH040-2 conferring high expression of the bla gene. Besides precisely processed beta-lactamase, transformed yeast cells contained mainly bla precursor up to the amount of 2% of total cellular protein. The precursor was shown to be synthesized on free polysomes in vivo but could be processed with rough microsomal membranes in a cell-free translation system. By applying an isolation procedure using high-salt conditions, the labile precursor could be separated in a native form from the mature beta-lactamase. Thereby it could be shown that the pre-beta-lactamase had virtually no enzymatic activity in contrast to the mature enzyme, which was indistinguishable from bacterial beta-lactamase. Furthermore, the precursor was highly susceptible to proteolytic degradation by trypsin under conditions which did not affect the mature enzyme. Accordingly, the protein conformation of the precursor must be substantially different from that of the authentic beta-lactamase, demonstrating that specific processing and transport of beta-lactamase is associated with directing the protein to a distinct conformation.
mesh_terms: D000818:Animals; D002474:Cell-Free System; D004591:Electrophoresis, Polyacrylamide Gel; D004792:Enzyme Precursors / Q000096:biosynthesis / Q000378:metabolism*; D008861:Microsomes / Q000378:metabolism; D009154:Mutation; D010447:Peptide Hydrolases / Q000378:metabolism; D011132:Polyribosomes / Q000378:metabolism; D014176:Protein Biosynthesis; D011487:Protein Conformation; D011817:Rabbits; D012441:Saccharomyces cerevisiae / Q000201:enzymology* / Q000235:genetics; D014170:Transformation, Genetic; D014357:Trypsin / Q000378:metabolism; D001618:beta-Lactamases / Q000096:biosynthesis / Q000378:metabolism*
chemical_list: D004792:Enzyme Precursors; D010447:Peptide Hydrolases; D014357:Trypsin; D001618:beta-Lactamases