Bacterial phosphotriesterase (PTE) catalyzes the hydrolysis of a wide variety of organophosphate nerve agents and insecticides. Previous kinetic studies with a series of enantiomeric organophosphate triesters have shown that the wild type PTE generally prefers the S(P)-enantiomer over the corresponding R(P)-enantiomers by factors ranging from 1 to 90. The three-dimensional crystal structure of PTE with a bound substrate analogue has led to the identification of three hydrophobic binding pockets. To delineate the factors that govern the reactivity and stereoselectivity of PTE, the dimensions of these three subsites have been systematically altered by site-directed mutagenesis of Cys-59, Gly-60, Ser-61, Ile-106, Trp-131, Phe-132, His-254, His-257, Leu-271, Leu-303, Phe-306, Ser-308, Tyr-309, and Met-317. These studies have shown that substitution of Gly-60 with an alanine within the small subsite dramatically decreased k(cat) and k(cat)/K(a) for the R(P)-enantiomers, but had little influence on the kinetic constants for the S(P)-enantiomers of the chiral substrates. As a result, the chiral preference for the S(P)-enantiomers was greatly enhanced. For example, the value of k(cat)/K(a) with the mutant G60A for the S(P)-enantiomer of methyl phenyl p-nitrophenyl phosphate was 13000-fold greater than that for the corresponding R(P)-enantiomer. The mutation of I106, F132, or S308 to an alanine residue, which enlarges the small or leaving group subsites, caused a significant reduction in the enantiomeric preference for the S(P)-enantiomers, due to selective increases in the reaction rates for the R(P)-enantiomers. Enlargement of the large subsite by the construction of an H254A, H257A, L271A, or M317A mutant had a relatively small effect on k(cat)/K(a) for either the R(P)- or S(P)-enantiomers and thus had little effect on the overall stereoselectivity. These studies demonstrate that by modifying specific residues located within the active site of PTE, it is possible to dramatically alter the stereoselectivity and overall reactivity of the native enzyme toward chiral substrates.

The factors that govern the substrate reactivity and stereoselectivity of phosphotriesterase (PTE) toward organophosphotriesters containing various combinations of methyl, ethyl, isopropyl, and phenyl substituents at the phosphorus center were determined by systematic alterations in the dimensions of the active site. The wild type PTE prefers the S(P)-enantiomers over the corresponding R(P)-enantiomers by factors ranging from 10 to 90. Enlargement of the small subsite of PTE with the substitution of glycine and alanine residues for Ile-106, Phe-132, and/or Ser-308 resulted in significant improvements in k(cat)/K(a) for the R(P)-enantiomers of up to 2700-fold but had little effect on k(cat)/K(a) for the corresponding S(P)-enantiomers. The kinetic preferences for the S(P)-enantiomers were thus relaxed without sacrificing the inherent catalytic activity of the wild type enzyme. A reduction in the size of the large subsite with the mutant H257Y resulted in a reduction in k(cat)/K(a) for the S(P)-enantiomers, while the values of k(cat)/K(a) for the R(P)-enantiomers were essentially unchanged. The initial stereoselectivity observed with the wild type enzyme toward the chiral substrate library was significantly reduced with the H257Y mutant. Simultaneous alternations in the sizes of the large and small subsites resulted in the complete reversal of the chiral specificity. With this series of mutants, the R(P)-enantiomers were preferred as substrates over the corresponding S(P)-enantiomers by up to 500-fold. These results have demonstrated that the stereochemical determinants for substrate hydrolysis by PTE can be systematically altered through a rational reconstruction of the dimensions of the active site.

Circadian clock and cell division cycle are two fundamental biological processes. The circadian clock is the body's molecular time-keeping system, while the cell division cycle regulates development and cellular renewal. The expression of cell cycle genes such as Wee1, Cyclins, and c-Myc are under circadian control and could be directly under the regulation of the circadian transcriptional complex. This complex is composed of heterodimer transactivators CLOCK/NPAS2 with BMAL1, which regulate the transcription of PER1, PER2, CRY1, and CRY2. In turn, the repressors CRY1 and CRY2 turn off the gene expressions of Per1/Per2, Cry1/Cry2 in a periodic manner by acting on the transcriptional complex. Two of these circadian rhythm regulators, PER1 and PER2, have now been linked to DNA damage response pathways in a series of papers that examined gene dosage. Overexpression of either Per1 or Per2 in cancer cells inhibits their neoplastic growth and increases their apoptotic rate. In vivo studies showed that mice deficient in mPer2 showed significant higher incidences of tumor development after genotoxic stress. Loss and dysregulation of Per1 and Per2 gene expression have been found in many types of human cancers. Recent studies demonstrate that both PER1 and PER2 are involved in ATM-Chk1/Chk2 DNA damage response pathways and implicate normal circadian function as a factor in tumor suppression.

Studies have suggested that the clock regulator PER2 is a tumour suppressor. A cancer network involving PER2 raises the possibility that some tumour suppressors are directly involved in the mammalian clock. Here, we show that the tumour suppressor promyelocytic leukaemia (PML) protein is a circadian clock regulator and can physically interact with PER2. In the suprachiasmatic nucleus (SCN), PML expression and PML-PER2 interaction are under clock control. Loss of PML disrupts and dampens the expression of clock regulators Per2, Per1, Cry1, Bmal1 and Npas2. In the presence of PML and PER2, BMAL1/CLOCK-mediated transcription is enhanced. In Pml À/À SCN and mouse embryo fibroblast cells, the cellular distribution of PER2 is primarily perinuclear/ cytoplasmic. PML is acetylated at K487 and its deacetylation by SIRT1 promotes PML control of PER2 nuclear localization. The circadian period of Pml À/À mice displays reduced precision and stability consistent with PML having a role in the mammalian clock mechanism.

We examined the kinetics of G␣ s and G␣ i regulation of human type V and type VI adenylyl cyclase (AC V and AC VI) in order to better model interactions between AC and its regulators. Activation of AC VI by G␣ s displayed classical Michaelis-Menten kinetics, whereas AC V activation by G␣ s was cooperative with a Hill coefficient of 1.4. The basal activity of human AC V, but not that of AC VI, was inhibited by G␣ i . Both enzymes showed greater inhibition by G␣ i at low G␣ s concentrations; however, human AC V was activated by G␣ i at high G␣ s concentrations. Neither regulator had an effect on the K m for Mg-ATP. Mutations made within the G␣ s binding pocket of AC V (N1090D) and VI (F1078S) displayed 6-and 14-fold greater EC 50 values for G␣ s activation but had no effect on G␣ i inhibition of basal activity or K m for Mg-ATP. G␣ s -stimulated AC VI-F1078S was not significantly inhibited by G␣ i , despite normal inhibition by G␣ i upon forskolin stimulation. Mechanistic models for G␣ s and G␣ i regulation of AC V and VI were derived to describe these results. Our models are consistent with previous studies, predicting a decrease in affinity of G␣ i in the presence of G␣ s . For AC VI, G␣ s is required for inhibition but not binding by G␣ i . For AC V, binding of two molecules of G␣ s and G␣ i to an AC dimer are required to fully describe the data. These models highlight the differences between AC V and VI and the complex interactions with two important regulators.The key to regulation of adenylyl cyclase (AC) 1 activity is the conformational state of the enzyme at the interface of the two large cytoplasmic domains (1-3). G␣ s binds to the second cytoplasmic (C 2 ) domain of AC and increases the affinity of the two domains for one another to promote catalysis (4 -8). G␣ i works in direct opposition to G␣ s , binding to the first cytoplasmic domain (C 1 ) and decreasing domain interaction to reduce formation of the AC catalytic site (3, 9).Although conformational changes at the domain interface are critical for regulation, other regions of AC play important roles as well. For example, the N terminus of rat AC VI interacts with the C 1 domain to modulate G␣ i -mediated inhibition (10) and glycosylation of AC VI may alter regulatory properties of the enzyme (11). Although native and recombinant G␣ s have similar effects on the cytoplasmic domains of AC, activation of full-length AC is less effectively inhibited by G␣ i upon stimulation of native versus recombinant G␣ s (12). Kleuss and Krause speculate that the palmitate on native G␣ s competes with the myristate on G␣ i for a hydrophobic binding pocket on AC (12). Previous studies by Taussig et al. (13) suggest that both G␣ s and G␣ i are bound simultaneously to the enzyme, seemingly in contradiction to studies using the independently expressed cytoplasmic domains (3). In an attempt to fully understand the regulation of full-length human AC V and VI, we have modeled the kinetics of G␣ s /G␣ i regulation.Types V and VI AC are generally grouped together as being hig...