The ability to use conformational flexibility is a hallmark of enzyme function. Here we show that protein motions and catalytic activity in a RNase are coupled and display identical solvent isotope effects. Solution NMR relaxation experiments identify a cluster of residues, some distant from the active site, that are integral to this motion. These studies implicate a single residue, histidine-48, as the key modulator in coupling protein motion with enzyme function. Mutation of H48 to alanine results in loss of protein motion in the isotope-sensitive region of the enzyme. In addition, kcat decreases for this mutant and the kinetic solvent isotope effect on kcat, which was 2.0 in WT, is near unity in H48A. Despite being located 18 Å from the enzyme active site, H48 is essential in coordinating the motions involved in the rate-limiting enzymatic step. These studies have identified, of Ϸ160 potential exchangeable protons, a single site that is integral in the rate-limiting step in RNase A enzyme function.Carr-Purcell-Meiboom-Gill dispersion ͉ enzyme dynamics ͉ NMR ͉ protein motions ͉ RNase A C onformational motions in enzymes play an essential role in their function and are often the rate-limiting step to overall catalytic throughput (1-5). Many enzymes have sufficiently evolved such that the bond-making and -breaking steps are fast relative to the ability of the enzyme to undergo a conformational change, and, thus, steps other than the chemical transformation of substrate are rate-limiting (ref. 6 and references therein). In systems such as these, understanding enzyme function requires characterization of the relevant time-dependent protein fluctuations from the timeaveraged three-dimensional structure. The ability of solution NMR spectroscopy to detect, with atomic resolution, motions over a wide timescale (picoseconds to seconds) makes it an ideal experimental technique to characterize conformational motions in proteins that can ultimately impact drug design, de novo enzyme construction, and enzyme engineering. In particular, relaxation-compensated Carr-Purcell-Meiboom-Gill (rcCPMG) dispersion experiments (7) are capable of informing on the kinetics, thermodynamics, and structural changes of protein motions in the microsecond-tomillisecond timescale.RNase A is an enzyme example in which a conformational change is the bottleneck to overall conversion of substrate to product (see ref. 3 for a review). RNase A catalyzes the cleavage of single-stranded RNA and does not require metal ions or cofactors. This enzyme has been studied in great detail as a model for protein folding, structure, and stability (8, 9). In addition, homologs of RNase A have important cytotoxic and antitumor properties (10). The rate-limiting step for the RNase A reaction is a protein conformational change that is coupled to the product release step (1, 11). This conformational change involves multiple amino acid residues throughout the protein, including those distant from the active site (12-14). These mobile regions include two loops: loop 1,...

In many cancers, high proliferation rates correlate with elevation of rRNA and tRNA levels, and nucleolar hypertrophy. However, the underlying mechanisms linking increased nucleolar transcription and tumorigenesis are only minimally understood. Here we show that IMP dehydrogenase-2 (IMPDH2), the rate-limiting enzyme for de novo guanine nucleotide biosynthesis, is overexpressed in the highly lethal brain cancer, glioblastoma (GBM). This leads to increased rRNA and tRNA synthesis, stabilization of the nucleolar GTP-binding protein, Nucleostemin, and enlarged, malformed nucleoli. Pharmacological or genetic inactivation of IMPDH2 in GBM reverses these effects and inhibits cell proliferation, whereas untransformed glia cells are unaffected by similar IMPDH2 perturbations. Impairment of IMPDH2 activity triggers nucleolar stress and growth arrest of GBM cells even in the absence of functional p53. Our results reveal that upregulation of IMPDH2 is a prerequisite for aberrant nucleolar function and increased anabolic processes in GBM, which constitutes a primary event in gliomagenesis.

The annonaceous acetogenins are the most potent of the known inhibitors of bovine heart mitochondrial complex I. These inhibitors act, at the terminal electron transfer step of the enzyme, in a similar way to the usual complex I inhibitors, such as piericidin A and rotenone; however, structural similarities are not apparent between the acetogenins and these known complex I inhibitors. A systematic set of isolated natural acetogenins was prepared and examined for their inhibitory actions with bovine heart mitochondrial complex I to identify the essential structural factors of these inhibitors for the exhibition of potent activity. Despite their very potent activity, the structural requirements of the acetogenins are not particularly rigid and remain somewhat ambiguous. The most common structural units, such as adjacent bis-tetrahydrofuran (THF) rings and hydroxyl groups in the 4- and/or 10-positions, were not essential for exhibiting potent activity. The stereochemistry surrounding the THF rings, surprisingly, seemed to be unimportant, which was corroborated by an exhaustive conformational space search analysis, indicating that the model compounds, with different stereochemical arrangements around the THF moieties, were in fairly good superimposition. Proper length and flexibility of the alkyl spacer moiety, which links the THF and the alpha, beta-unsaturated gamma-lactone ring moieties, were essential for the potent activity. This probably results from some sort of specific conformation of the spacer moiety which regulates the two ring moieties to locate into an optimal spatial position on the enzyme. It is, therefore, suggested that the structural specificity of the acetogenins, required for optimum inhibition, differs significantly from that of the common complex I inhibitors in which essential structural units are compactly arranged and conveniently defined. The structure-activity profile for complex I inhibition is discussed in comparison with those for other biological activities.

A full-length cDNA for the rat kidney mitochondrial cytochrome P450 mixed function oxidase, 25-hydroxyvitamin D 3 -1␣-hydroxylase (P4501␣), was cloned from a vitamin D-deficient rat kidney cDNA library and subcloned into the mammalian expression vector pcDNA 3.1(؉). When P4501␣ cDNA was transfected into COS-7 transformed monkey kidney cells, they expressed 25-hydroxyvitamin D 3 -1␣-hydroxylase activity. The sequence analysis showed that P4501␣ was of 2,469 bp long and contained an ORF encoding 501 amino acids. The deduced amino acid sequence showed a 53% similarity and 44% identity to the vitamin D 3 -25-hydroxylase (CYP27), whereas it has 42.6% similarity and 34% identity with the 25-hydroxyvitamin D 3 -24-hydroxylase (CYP24). Thus, it composes a new subfamily of the CYP27 family. Further, it is more closely related to the CYP27 than to the CYP24. The expression of P4501␣ mRNA was greatly increased in the kidney of vitamin D-deficient rats. In rats with the enhanced renal production of 1␣,25-dihydroxyvitamin D 3 (rats fed a low Ca diet), P4501␣ mRNA was greatly increased in the renal proximal convoluted tubules. has been demonstrated to be the active form of vitamin D 3 (1, 2). The production of 1␣,25(OH) 2 D 3 is regulated primarily at the final step in its synthetic pathway; 1␣-hydroxylation of 25(OH)D 3 by the renal mitochondrial P450 mixed-function oxidase (3, 4). The cytochrome P450 enzymes form a superfamily of hemecontaining proteins that are bound to the membranes of the microsomes and mitochondria and serve as an oxidationreduction component of the mixed-function oxidase system. In in vivo conditions, this system is involved in the oxidative metabolism of a number of steroids. The CYP family is large, with at least 74 families, and each mammalian species is estimated to have between 60 and 200 distinct superfamily members (5, 6). These P450 proteins have more than 40% amino acid homology within a single family and usually more than 55% identity within a subfamily. The cysteine-containing region associated with the heme-binding domain of the P450 protein is highly conserved among all eukaryotic species.The amino acid sequence similarity between 25(OH)D 3 -24-hydroxylase (CYP24) and vitamin D 3 -25-hydroxylase (CYP27) enzymes within the heme-binding domain has been reported to be 60% (7,8). The homology of the putative ferredoxinbinding domain between CYP27 and CYP24 was 70%. By using a probe from the 3Ј region of the rat CYP24 cDNA, which encompasses the heme-binding domain of the molecule, St-Arnaud et al. (9) screened a cDNA library from the kidney of vitamin D-deficient rats under reduced stringency conditions to identify and clone the 25(OH)D 3 -1␣-hydroxylase cDNA. They reported the partial amino acid sequence of P4501␣ at the 18th Annual Meeting of the American Society for Bone and Mineral Research (September 9-11, 1996, Seattle, WA). The amino acid sequence similarity with CYP24 was calculated to be 78% within the heme-binding domain; the two proteins diverged significantly outside this r...