Putative domain--domain interactions of the monomeric bifunctional enzyme indoleglycerol phosphate synthase:phosphoribosyl anthranilate isomerase from Escherichia coli were probed by separating the domains on the gene level and expressing them as monofunctional proteins. The engineered monofunctional enzymes were found to be stable, monomeric proteins with virtually full catalytic activity. In addition, binding of indolyglycerol phosphate to the active site of indoleglycerol phosphate synthase and binding of reduced 1-[(2-carboxyphenyl)amino]-1-deoxyribulose 5-phosphate, a competitive inhibitor of both indoleglycerol phosphate synthase and phosphoribosyl anthranilate isomerase, were almost identical in both the mono- and bifunctional enzymes. Furthermore, no association between the monofunctional enzymes was found, neither in vitro, by sedimentation and gel filtration experiments, nor in vivo, by coexpression of the domains in the same cell. Thus, no selective advantages of the bifunctional enzyme from Escherichia coli over the respective monofunctional enzymes were found on a functional level. However, the phosphoribosyl anthranilate isomerase domain appears to stabilize the indoleglycerol phosphate synthase domain of the bifunctional enzyme from Escherichia coli by interactions that seem to subtly influence the kinetics of ligand binding.

The 12 January 2010 Mw 7.0 earthquake in the Republic of Haiti caused an estimated 300,000 deaths, displaced more than a million people, and damaged nearly half of all structures in the epicentral area. We provide an overview of the historical, seismological, geotechnical, structural, lifeline-related, and socioeconomic factors that contributed to the catastrophe. We also describe some of the many challenges that must be overcome to enable Haiti to recover from this event. Detailed analyses of these issues are presented in other papers in this volume.

Data from steady state and transient kinetics show that the functional phosphoribosyl anthranilate isomerase domain of the naturally bifunctional enzyme from Escherichia coli has properties similar to those of its artificially excised domain. The naturally monofunctional enzyme from Saccharomyces cerevisiae has significantly higher values of both kcat and kcat/KMPRA. The primary product of a single turnover of phosphoribosylanthranilate is fluorescent, but it slowly isomerizes to the nonfluorescent stable product. The latter is the competent substrate of indoleglycerol phosphate synthase, which catalyzes the subsequent step of tryptophan biosynthesis. The isomerization is characterized by a monoexponential decay independent of phosphoribosyl anthranilate isomerase. Due to a tentative assignment of the fluorescent, primary product and the nonfluorescent, stable product to an enol and a keto compound, respectively, tryptophan biosynthesis appears to be rate-limited by an uncatalyzed enol/keto tautomerization. A formal kinetic mechanism of the reaction catalyzed by phosphoribosyl anthranilate isomerase is proposed that is consistent with the combined enzymic and ligand binding properties of the three variants of phosphoribosyl anthranilate isomerase.

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Ca2+ and Mg2+ binding to rat parvalbumin was measured by means of the fluorescent Ca2+ indicator fluo‐3 using a method developed earlier [Eberhard, M. & Erne, P. (1991) Eur. J. Biochem. 202, 1333–1338]. We demonstrate that rat parvalbumin contains two equivalent Ca2+/Mg2+ binding sites and that Ca2+ and Mg2+ compete for the same sites. Dissociation constants (Kd) for Ca2+ and Mg2+ in Hepes buffer containing 150 mM K+ at 35°C and pH 7.2 are 11.0 ± 1.8 nM and 41 ± 8 μM, respectively. At an ionic strength below 0.2 M, Kd values of Ca2+ binding to rat parvalbumin are approximately proportional to the ion concentration. Kd values of Ca2+ binding were found to be about fourfold larger in the presence of Na+ as compared with K+, indicating that Na+ distinctly influences Ca2+ binding to rat parvalbumin. Both Ca2+ and Mg2+ binding to parvalbumin are exothermic whereas Ca2+ and Mg2+ binding to fluo‐3 are endothermic entropy‐driven processes.