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The aspartic acid residues at 273 and 275 are involved in the binding of a metal cation to NADP-dependent isocitrate dehydrogenase. The aspartic acid residues at 275 and 279 are required for catalytic activity.

A study was conducted to determine the catalytic properties of mutant alpha3beta3gamma subcomplexes of TF1-adenosine triphosphatase (ATPase) from Bacillus PS3 containing the alphaF244C, alphaY300C and alphaR304C substitutions. The subcomplexes were found out to have unique features for hydrolyzing ATP and forming inhibitory adenosine diphosphate-fluoroaluminate complexes in catalytic sites. Results suggested that the substitutions of TF1 may be part of a pathway communicating conformational signals from one catalytic site to another.

The rates of inactivation of the wild-type and mutant alpha3beta3gamma subcomplexes of TF1 in the presence of ADP, Mg2+, Al3+ and F- were investigated. The results showed a slow, but complete, inactivation when Al3+ and F- were added to wild type and mutan alpha3beta3gamma subcomplexes containing MgADP bound to a single catalytic site.

Temperature lowering and pyrophosphate (PPi) binding to noncatalytic sites of endogenous nucleotides (nd-MF1) promote the dissociation of inhibitory MgADP from a catalytic site, thereby regulating the ATPase activity of the beef heart mitochondrial F-ATPase. Kinetic properties of ATP hydrolysis vary with temperature and during temperatures above 18 degrees Celsius, the inhibitory MgADP is entrapped in the catalytic site of the enzyme. When noncatalytic sites are unoccupied, only F1-ATPases exhibit stimulation of ATPase activity by PPi.

The crystal structure of the catalytic domain of PKC[beta]II, the conventional protein kinase C isoform, complexed with a bisindolylmaleimide inhibitor is described. The pathogenic role of PKC[beta]II in the development of diabetic complications has shown that the structure can serve as a template for the rational design of inhibitors as potential therapeutic agents.

The crystal structure of procine heart mitochondrial NADP- dependent isocitrate dehydrogenase complexed with Mn(super 2+) and isocitrate, Asp(super 252), Asp(super 275) and Asp(super 279) is selected as targets for site-directed mutagenesis to determine the roles of these three residues as ligands of the metal ion. The study of site-directed mutagenesis of ligands of Mn(super 2+) bound to the procine mitochondrial NADP-dependent isocitrate dehydrogenase is consistent with the crystal structure of the enzyme-substrate complex.

The conventional protein kinase C isoform, PKCβII, is a signaling kinase activated during the hyperglycemic state and has been associated with the development of microvascular abnormalities associated with diabetes. PKCflII, therefore, has been identified as a therapeutic target where inhibitors of its kinase activity are being pursued for treatment of microvascular-related diabetic complications. In this report, we describe the crystal structure of the catalytic domain of PKCβII complexed with an inhibitor at 2.6 Å resolution. The kinase domain of PKCβII was cleaved and purified from full-length PKCβII expressed in baculovirus-infected insect cells. The overall kinase domain structure follows the classical bilobal fold and is in its fully activated conformation with three well-defined phosphorylated residues: Thr-500, Thr-641, and Ser-660. Different from the crystal structures of nonconventional PKC isoforms, the C-terminus of the PKCβII catalytic domain is almost fully ordered and features a novel α helix in the turn motif. An ATP-competitive inhibitor, 2-methyl-1H-indol-3-yl-BIM-1, was crystallized with the PKCβII catalytic domain as a dimer of two enzyme-inhibitor complexes. The bound inhibitor adopts a nonplanar conformation in the ATP-binding site, with the kinase domain taking on an intermediate, open conformation. This PKCβII-inhibitor complex represents the first structural description of any conventional PKC kinase domain. Given the pathogenic role of PKCβII in the development of diabetic complications, this structure can serve as a template for the rational design of inhibitors as potential therapeutic agents. [ABSTRACT FROM AUTHOR]

Pig heart mitochondrial NADP-dependent isocitrate dehydrogenase requires a divalent metal ion for catalysis, and metal-isocitrate is its preferred substrate. On the basis of the crystal structure of the enzyme-Mn2+-isocitrate complex, Asp252, Asp275, and Asp279 were selected as targets for site-directed mutagenesis to evaluate the roles of these residues as ligands of the metal ion. The circular dichroism spectra of the purified mutant enzymes are similar to that of wild-type enzyme indicating there are no appreciable conformational changes. The Km values for isocitrate and for Mn2+ are increased in the asparagine and histidine mutants at positions 252 and 275; while for cysteine mutants at the same positions, the Km's are not changed appreciably. Mutants at position 279 exhibit only a small change in Km for isocitrate. These results indicate that Asp252 and Asp275 are ligands of enzyme-bound Mn2+and influence the binding of Mn2+-isocitrate. Cysteine is an acceptable substitute for aspartate as a ligand of Mn2+. The pKaes's of D252C and D275C enzymes are similar to that of the wild-type enzyme (about 5.2), while the PKaes of D279C is a little lower (about 4.7). These findings suggest that the Vmax's of the D252C, D275C, and D279C enzymes depend on the ionizable form of the same group as in wild-type enzyme and neither Asp252, Asp275, nor Asp279 acts as the general base in the enzymatic reaction. For wild-type enzyme, the pKaes varies with the metal ion used with Mg2+ > Cd2+ > Mn2+ > Co2+, similar to the order of the pK's for these four metal-bound waters. [ABSTRACT FROM AUTHOR]

The bovine heart mitochondrial F1-ATPase (MF1) is reversibly inhibited in the dark by 4-amino-1-octylquinaldinium (AOQ) with an I0.5 value of 48 μ M. When irradiated in the presence of AOQ, MF1 is photoinactivated with an apparent Kd of 12 μ M. About 1.1 mol of [3H]AOQ were incorporated per mol of MF1 on complete photoinactivation. Fractionation of a cyanogen bromide digest of MF1 photolabeled with [3H]AOQ followed by fractionation of peptic digests of partially purified cyanogen bromide fragments led to isolation of two CNBr/peptic fragments labeled with3H. Sequence analysis of the labeled peptides revealed that one contained residues 423–441 of the β subunit. A gap in position 2 of the sequence indicates that βPhe424 is derivatized. The phenyl side-chain of this residue is part of a pocket that binds the adenine moiety of ATP or ADP at catalytic sites. The other peptide, which was labeled to a greater extent, contained residues 342–358 of the β subunit, but in this case, no gap was found in the sequence indicating that the derivatized amino-acid side-chain might not have survived the conditions of automatic Edman degradation. This peptide contains βTyr345, the side-chain of which is also a component of the pocket that binds the adenine moiety of ATP or ADP to catalytic sites. However, for the reason stated, there is no direct evidence that βTyr345 is labeled in this peptide. [ABSTRACT FROM AUTHOR]