ABSTRACT
Mn(2+)-assisted catalysis by B. stearothermophilus TrpRS parallels that in polymerases and reduces specificity in amino acid activation. As predicted by nonequilibrium molecular dynamics simulations, multivariant thermodynamic cycles with [ATP]-dependent Michaelis-Menten kinetics and Mn(2+) for Mg(2+) substitution demonstrate energetic coupling of ATP affinities to the metal; to lysines K111 and K192, which interact via the PPi leaving group; and to K195, which couples differently to the metal via the alpha-phosphate. However, net coupling to the metal opposes catalysis in both ground (K(M)) and transition (k(cat)) states. The 10(5)-fold rate acceleration by Mg(2+)-protein interactions therefore requires additional favorable protein-metal couplings. Examples include longer-range, i.e., allosteric, interactions previously illustrated by the remote F37I mutation, which both reduces k(cat) and enhances catalytic assist by Mn(2+), relative to that by Mg(2+). These data support a model linking metal-assisted phosphoryl transfer catalysis to domain movement, and hence to free-energy transduction in a broad range of enzymes.
Subject(s)
Geobacillus stearothermophilus/enzymology , Magnesium/chemistry , Tryptophan-tRNA Ligase/chemistry , Tryptophan-tRNA Ligase/metabolism , Allosteric Regulation , Binding Sites , Catalysis , Catalytic Domain , Crystallography, X-Ray , Escherichia coli/genetics , Geobacillus stearothermophilus/metabolism , Kinetics , Lysine/chemistry , Magnesium/metabolism , Metals/chemistry , Models, Chemical , Models, Molecular , Mutation , Protein Binding , Protein Conformation , Protein Structure, Tertiary , Thermodynamics , Tryptophan-tRNA Ligase/geneticsABSTRACT
BP1, a homeobox gene, is overexpressed in the bone marrow of 63% of acute myeloid leukemia patients. In this study, we compared the growth-inhibitory and cyto-differentiating activities of all-trans retinoic acid (ATRA) in NB4 (ATRA-responsive) and R4 (ATRA-resistant) acute promyelocytic leukemia (APL) cells relative to BP1 levels. Expression of two oncogenes, bcl-2 and c-myc, was also assessed. NB4 and R4 cells express BP1, bcl-2, and c-myc; the expression of all three genes was repressed after ATRA treatment of NB4 cells but not R4 cells. To determine whether BP1 overexpression affects sensitivity to ATRA, NB4 cells were transfected with a BP1-expressing plasmid and treated with ATRA. In cells overexpressing BP1: (1) proliferation was no longer inhibited; (2) differentiation was reduced two- to threefold; (3) c-myc was no longer repressed. These and other data suggest that BP1 may regulate bcl-2 and c-myc expression. Clinically, BP1 levels were elevated in all pretreatment APL patients tested, while BP1 expression was decreased in 91% of patients after combined ATRA and chemotherapy treatment. Two patients underwent disease relapse during follow-up; one patient exhibited a 42-fold increase in BP1 expression, while the other showed no change. This suggests that BP1 may be part of a pathway involved in resistance to therapy. Taken together, our data suggest that BP1 is a potential therapeutic target in APL.
