Larry D. Faller

Inferences about the catalytic domain of P-type ATPases from the tertiary structures of enzymes that catalyze the same elementary reaction

The machinery to catalyze elementary reactions is conserved, and the number of solved enzyme structures is increasing exponentially. Therefore, structures of enzymes that catalyze phosphate transfer are reviewed, and a supersecondary structure connecting the Walker A sequence to another sequence containing functional amino acids is proposed as an additional signature for the active site. The new signature is used to infer the identity of the P‐loop in P‐type biological pumps and may be useful in predicting targets for site‐directed mutagenesis in other enzymes of unknown structure like the AAA family and ABC transporters.

Site-Directed Mutagenesis of Amino Acids in the Cytoplasmic Loop 6/7 of Na,K-ATPase

Abstract: The loop between transmembrane helices 6 and 7 (L6/7) of P‐type ATPases has been suggested to be important for the functional linkage of ion binding and enzyme phosphorylation or to be a site of initial cation binding. To investigate the role of L6/7 in Na,K‐ATPase, alanine substitutions were made for charged and conserved residues in L6/7 of the human α1 subunit and the proteins were expressed in yeast for analysis. All mutants except the triple mutant E825A/E828A/D830A bound ouabain. Although the equilibrium dissociation constant for ouabain binding by most mutants was similar to the wild‐type value, the Kd of R837A for ouabain binding was ∼15‐fold higher than the wild‐type Kd. 18O exchange measurements indicated that the apparent affinity of this mutant for Pi was reduced about 3‐fold. The concentration dependence of KCl inhibition of ouabain binding or of NaCl inhibition of ouabain binding revealed 2–4‐fold changes in the apparent affinity for cations in the E825A, E828A, and R837A mutants. The E825A and E828A mutants lost the ability to bind ouabain after extraction with 0.1% SDS or after brief heating, indicating that these mutations affected the stability of the enzyme. The ATPase activity of the other mutants was measured after extraction of crude yeast membranes with 0.1% SDS. For all mutants except R834A, R837A, and R848A, the activity was at least 50% of wild‐type activity.

Mechanism of the Conformational Change in Fluorescein 5′-Isothiocyanate-Modified Na+/K+ ATPase

It is generally agreed that conformational changes explain both coupling of transport to ATP hydrolysis (16) and the physical translocation of Na+ and K+ ions by sodium pump (12). Karlish reported 15 years ago at the IInd Annual Conference on the Sodium Pump (10) that a conformational change can be studied by chemically modifying the enzyme with fluorescein S′-isothiocyanate (FITC). He showed that the time course of the reaction can be followed with a stopped-flow instrument, and he proposed that the reaction reported by fluorescein is a rate-limiting conformational change in dephosphoenzyme (11). Subsequent studies of the conformational change in dephosphoenzyme using fluorescein and other reporter groups have been reviewed by Glynn (6).