Abstract：
:An alanine racemase encoded by a gene from the thermophilic Gram-positive bacterium Bacillus stearothermophilus is overproduced to 0.3% of the soluble protein when carried on plasmid pICR4 in Escherichia coli [Inagaki, K., Tanizawa, K., Badet, B., Walsh, C. T., Tanaka, H., & Soda, K. (1986) Biochemistry (third paper of four in this issue)]. Purification of large quantities (50 mg) of racemase permits study of time-dependent inactivation by D and L isomers of the antibacterial (1-aminoethyl)phosphonate (Ala-P), the phosphonate analogue of alanine. The time-dependent activity loss by this compound now appears general to Gram-positive but not to Gram-negative racemases [Badet, B., & Walsh, C. (1985) Biochemistry 24, 1333] and is shown to occur by extremely slow dissociation of a noncovalent E X Ala-P complex. Ala-P binds initially in a weak, reversible (KI = 1 mM) competitive manner but is slowly isomerized (kinact = 6-9 min-1) to a stoichiometric enzyme complex, which in turn dissociates extremely slowly, with a half-time about 25 days. Thus, Ala-P is a slow but not a tight-binding inhibitor. The E X Ala-P complex is not reducible by borohydride but does perturb the fluorescence of bound pyridoxal 5'-phosphate coenzyme. Determination of the sequence of an active site octapeptide of the B. stearothermophilus alanine racemase shows homology with the sequence of a Gram-negative Salmonella typhimurium alanine racemase that is not susceptible to time-dependent inhibition by Ala-P. Studies with Ala-P analogues suggest the phosphonate dianion is crucial for stable formation of an isomerized long-lived E X Ala-P-inhibited complex.
journal_name
Biochemistryjournal_title
Biochemistryauthors
Badet B,Inagaki K,Soda K,Walsh CTdoi
10.1021/bi00359a029subject
Has Abstractpub_date
1986-06-03 00:00:00pages
3275-82issue
11eissn
0006-2960issn
1520-4995journal_volume
25pub_type
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