Adenylate kinase from Bacillus subtilis, like the enzyme from Bacillus stearothermophilus, contains a structural zinc atom. Cys153 in the enzyme from B. stearothermophilus, which is involved in the zinc coordination, is replaced in the adenylate kinase from B. subtilis by an aspartic acid residue. Therefore, we were interested in establishing whether this difference has an impact on the structure, the metal chelation, and the overall stability of these proteins. We also were interested in determining whether His138, which is conserved in many adenylate kinases, can act as a fourth partner in the metal chelation and, in general, whether His can successfully replace Cys or Asp in coordinating zinc in the adenylate kinase from B. subtilis. The adk gene from B. subtilis was cloned by polymerase chain reaction. The wild-type protein, together with several variants obtained by site-directed mutagenesis, were expressed in Escherichia coli and analyzed by biochemical and physicochemical methods. The H138N and D153C mutants of adenylate kinase from B. subtilis exhibited properties similar to those of the wild-type protein, indicating that His138 is not involved in metal coordination and that Asp153, just like Cys in the analogous position in the enzyme from B. stearothermophilus, can participate in zinc chelation. This is the first experimental evidence indicating that aspartic acid can be involved in the coordination of a structural zinc atom. On the other hand, the D153H and D153T variants showed significant changes in their zinc-binding properties. Dialysis of the latter proteins against buffer (in both the presence and the absence of 2 mM EDTA) resulted in removal of the metal ion and loss of enzymatic activity.(ABSTRACT TRUNCATED AT 250 WORDS)