This article describes the design, synthesis and characterization of an optical sensor suitable for practical measurement of ionized calcium in serum and whole blood samples. The key to the development of this sensor is the identification of a chemically very stable, nitrogen-containing, calcium selective ionophore, coupled with a fluorophore having the correct spectral and electron accepting p...

The most common procedure in searching for active sites in biological macromolecules is partial hydrolysis. In a few cases, acidic, alkaline, or enzymatic breakdown of the macromolecule results in the removal of the inert sites of the complex, reducing the size of the remainder to the active core structure. Similar treatments frequently serve as steps of purification by removing noncovalently b...

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An emerging paradigm in enzymology is that transient high-energy structural states play crucial roles in enzymatic reaction cycles. Generally, these high-energy or 'invisible' states cannot be studied directly at atomic resolution using existing structural and spectroscopic techniques owing to their low populations or short residence times. Here we report the direct NMR-based detection of the m...

Elucidating the origin of enzymatic catalysis stands as one the great challenges of contemporary biochemistry and biophysics. The recent emergence of computational enzymology has enhanced our atomistic-level description of biocatalysis as well the kinetic and thermodynamic properties of their mechanisms. There exists a diversity of computational methods allowing the investigation of specific en...

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My understanding is that “reflections” can cover a multitude of sins. I have previously written a brief history of my career, and do not want to duplicate that exposition (1). Instead, this “reflection” will trace a primary focus of my laboratory for many decades, namely how do enzymes achieve their remarkable catalytic efficiency? (Indeed this has been a focal point of the field of biochemistr...

The glpX gene from Francisella tularensis encodes for the class II fructose 1,6-bisphosphatase (FBPaseII) enzyme. The glpX gene has been verified to be essential in F. tularensis, and the inactivation of this gene leads to impaired bacterial growth on gluconeogenic substrates. In the present work, we have complemented a ∆glpX mutant of Escherichia coli with the glpX gene of F. tularensis (FTF16...

Robin van der Lee,*,†,‡ Marija Buljan,†,▲ Benjamin Lang,†,▲ Robert J. Weatheritt,†,▲ Gary W. Daughdrill, A. Keith Dunker, Monika Fuxreiter, Julian Gough, Joerg Gsponer, David T. Jones, Philip M. Kim, Richard W. Kriwacki, Christopher J. Oldfield, Rohit V. Pappu, Peter Tompa, Vladimir N. Uversky, Peter E. Wright, and M. Madan Babu*,† †MRC Laboratory of Molecular Biology, Francis Crick Avenue, Cam...

Enzyme inhibition due to the reversible binding of reaction products is common and underlies the origins of negative feedback inhibition in many metabolic and signaling pathways. Product inhibition generates non-linearity in steady-state time courses of enzyme activity, which limits the utility of well-established enzymology approaches developed under the assumption of irreversible product rele...