Remote mutations alter transition-state structure of human purine nucleoside phosphorylase

Purine nucleoside phosphorylase (PNP) catalyzes the reversible phosphorolysis of purine (2'-deoxy)ribonucleosides to give the corresponding purine base and (2'-deoxy)ribose 1-phosphate as products. Human and bovine PNPs (HsPNP and BtPNP) form distinct transition states despite 87% identity in amino acid sequence. A PNP hybrid was produced by replacing K22 and H104 in HsPNP with the corresponding Glu and Arg residues found in BtPNP. We solved the transition-state structure of E:R-HsPNP (K22E:H104R mutant of HsPNP) using competitive kinetic isotope effects (KIE) and global density functional calculations. An array of PNP transition states was generated from optimized structure candidates with varied C1'-N9, C1'-Ophosphate distances, ribosyl pucker configurations and N7-protonation states. Isotopically labeled [1'-3H], [2'-3H], [1'-14C], [9-15N], [1'-14C, 9-15N] and [5'-3H2]inosines gave intrinsic KIE values of 1.210, 1.075, 1.035, 1.024, 1.065, 1.063 with E:R-HsPNP, respectively. The suite of E:R-HsPNP KIEs match a single structure from the array of PNP transition-state candidates. The transition state of E:R-HsPNP is fully dissociative, N7-protonated hypoxanthine (C1'-N9 distance >or= 3.0 A) with partial participation of phosphate (C1'-Ophosphate distance = 2.26 A), 2'-C-exo-ribosyl ring pucker and the O5'-C5'-C4'-O4' dihedral angle near 60 degrees . The transition state of E:R-HsPNP is altered from the fully dissociative DN*AN character for HsPNP to a late phosphate-associative character. E:R-HsPNP differs from native HsPNP by only two residues over 25 A away from the active site. New interactions caused by the mutations increase the catalytic efficiency of the enzyme for formation of a late transition state with increased participation of the phosphate nucleophile. Dynamic coupling motions from the remote mutations to the catalytic sites are proposed.     

Second-sphere amino acids contribute to transition-state structure in bovine purine nucleoside phosphorylase

Transition-state structures of human and bovine of purine nucleoside phosphorylases differ, despite 87% homologous amino acid sequences. Human PNP (HsPNP) has a fully dissociated transition state, while that for bovine PNP (BtPNP) has early SN1 character. Crystal structures and sequence alignment indicate that the active sites of these enzymes are the same within crystallographic analysis, but residues in the second-sphere from the active sites differ significantly. Residues in BtPNP have been mutated toward HsPNP, resulting in double (Asn123Lys; Arg210Gln) and triple mutant PNPs (Val39Thr; Asn123Lys; Arg210Gln). Steady-state kinetic studies indicated unchanged catalytic activity, while pre-steady-state studies indicate that the chemical step is slower in the triple mutant. The mutant enzymes have higher affinity for inhibitors that are mimics of a late dissociative transition state. Kinetic isotope effects (KIEs) and computational chemistry were used to identify the transition-state structure of the triple mutant. Intrinsic KIEs from [1'-3H], [1'-14C], [2'-3H], [5'-3H], and [9-15N] inosines were 1.221, 1.035, 1.073, 1.062 and 1.025, respectively. The primary intrinsic [1'-14C] and [9-15N] KIEs indicate a highly dissociative SN1 transition state with low bond order to the leaving group, a transition state different from the native enzyme. The [1'-14C] KIE suggests significant nucleophilic participation at the transition state. The transition-state structure of triple mutant PNP is altered as a consequence of the amino acids in the second sphere from the catalytic site. These residues are implicated in linking the dynamic motion of the protein to formation of the transition state.     

Picomolar inhibitors as transition-state probes of 5'-methylthioadenosine nucleosidases.

Transition states can be predicted from an enzyme's affinity to related transition-state analogues. 5'-Methylthioadenosine nucleosidases (MTANs) are involved in bacterial quorum sensing pathways and thus are targets for antibacterial drug design. The transition-state characteristics of six MTANs are compared by analyzing dissociation constants (K(d)) with a small array of representative transition-state analogues. These inhibitors mimic early or late dissociative transition states with K(d) values in the picomolar range. Our results indicate that the K(d) ratio for mimics of early and late transition states are useful in distinguishing between these states. By this criterion, the transition states of Neisseria meningitides and Helicobacter pylori MTANs are early dissociative, whereas Escherichia coli, Staphylococcus aureus, Streptococcus pneumoniae, and Klebsiella pneumoniae MTANs have late dissociative characters. This conclusion is confirmed independently by the characteristic [1'- (3)H] and [1'- (14)C] kinetic isotope effects (KIEs) of these enzymes. Large [1'- (3)H] and unity [1'- (14)C] KIEs are observed for late dissociative transition states, whereas early dissociative states showed close-to-unity [1'- (3)H] and significant [1'- (14)C] KIEs. K d values of various MTANs for individual transition-state analogues provide tentative information about transition-state structures due to varying catalytic efficiencies of enzymes. Comparing K d ratios for mimics of early and late transition states removes limitations inherent to the enzyme and provides a better predictive tool in discriminating between possible transition-state structures.     

Fluorinated cyclitols as useful biological probes of phosphatidylinositol metabolism

A number of deoxyfluoro cyclitols have been synthesized and evaluated as probes of the phosphatidylinositol pathway (PtdIns pathway), most notably 5-deoxy-5-fluoro-myo-inositol, which is incorporated into the pathway at about 25% the level of myo-inositol itself. Unfortunately, none of the cyclitols have proved effective in limiting cell proliferation, as the cells are able to 'synthesize around' the fraudulent cyclitols using natural myo-inositol as substrate. Inhibitors for 3-phosphatidylinositol kinase, which has importance in a number of pathological conditions, including cancer, have been intensively investigated. 3-Deoxy-3-fluoro-myo-inositol is incorporated into the PtdIns pathway; however, only related phosphatidyl derivatives, for example, a methyl ether derivative of the 3-deoxy inositol, showed significant antiproliferative activity. Synthesis of the deoxyfluoro analogues most often has been accomplished by DAST fluoro-de-hydroxylation of the appropriate cyclitol, generally leading to products of inversion.     

The three-dimensional structure of acarbose bound to glycogen phosphorylase

Acarbose, a pseudotetrasaccharide with a conduritol ring at the nonreducing terminus, is a naturally occurring inhibitor of amylases. It is shown here to be an inhibitor of glycogen phosphorylase and to bind more tightly to the enzyme than the equivalent malto-oligosaccharide substrate. X-ray crystallographic studies of the acarbose-phosphorylase a complex in the presence of glucose and caffeine reveal the structure of acarbose as bound to the storage site of phosphorylase. The acarbose binds in an orientation such that the conduritol ring makes no protein contacts. As with malto-oligosaccharides bound at this site, the observed conformation of acarbose is stabilized by O-2-O-3' hydrogen bonding and is similar to, but not identical with, that predicted by hard-sphere exo-anomeric effect calculations and justified by 1H nuclear magnetic resonance studies (Bock, K., and Pedersen, H. (1984) Carbohydr. Res. 132, 142-149). Intramolecular O2-O3' hydrogen bonds appear to play an important role in stabilizing the conformation observed in these studies, even for those residues closely associated with the protein.     

Fluorinated analogues of spermidine as substrates of spermine synthase

A series of mono- and geminal difluorinated analogues of spermidine (4-azaoctane-1,8-diamine) have been tested as potential substrates of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase (EC 2.5.1.22). Substitution of the hydrogen atoms of the methylene group at position 7 by one or two fluorine atoms decreases 8-fold and 160-fold the apparent Km values for the HTC cell enzyme respectively. Similarly, the Km values of 7-monofluoro and 7,7-difluorospermidine for the pure bovine enzyme are reduced 8-fold and 100-fold respectively, in comparison with spermidine. Di-, but not monofluoro substitution, in the 6-position causes a 5-fold reduction in the affinity for the HTC cell enzyme. Gem-fluorine substitution in the 2-position abolishes substrate capability. In addition to their high affinity for spermine synthase, 7-monofluorospermidine and 7,7-difluorospermidine cause substrate inhibition. This phenomenon, which is more pronounced in the case of the difluorinated analogues is pH-dependent. These enzymatic findings are discussed with regard to the protonation sites of the spermidine analogues, determined by potentiometric titration, which vary as a function of the number and position of the fluorine substituents relative to the basic amino groups.     

Interaction between glycogen and glycogen phosphorylase of Tetrahymena

The glycogen phosphorylase of Tetrahymena pyriformis complexes with glycogen as judged by its elution pattern from columns of Sepharose 6B. Complex formation does not occur with starch, amylose, or amylopectin, and neither do these polyglucans serve as primers for the enzyme. To study the association between the phosphorylase and glycogen particles in situ, Tetrahymena were grown under differing physiological conditions, phosphorylase was isolated and chromatographed on a Sepharose 6B column. Phosphorylase activity isolated from cells grown in the absence of glucose was only partially associated with glycogen, while in cells exposed to glucose for 30 min or more all the phosphorylase activity was associated with glycogen. The effects of culture age and anaerobiosis on the relative amounts of free and glycogen-bound enzyme in the cells were also studied. It was concluded from the in vivo experiments that there was no simple relation between the fraction of enzyme bound to glycogen and between cell glycogen content.     

Three-dimensional structure of phosphorylase kinase at 22 A resolution and its complex with glycogen phosphorylase b.

Phosphorylase kinase (PhK) integrates hormonal and neuronal signals and is a key enzyme in the control of glycogen metabolism. PhK is one of the largest of the protein kinases and is composed of four types of subunit, with stoichiometry (alphabetagammadelta)(4) and a total MW of 1.3 x 10(6). PhK catalyzes the phosphorylation of inactive glycogen phosphorylase b (GPb), resulting in the formation of active glycogen phosphorylase a (GPa) and the stimulation of glycogenolysis. We have determined the three-dimensional structure of PhK at 22 A resolution by electron microscopy with the random conical tilt method. We have also determined the structure of PhK decorated with GPb at 28 A resolution. GPb is bound toward the ends of each of the lobes with an apparent stoichiometry of four GPb dimers per (alphabetagammadelta)(4) PhK. The PhK/GPb model provides an explanation for the formation of hybrid GPab intermediates in the PhK-catalyzed phosphorylation of GPb.     

Interaction of calmodulin and glycogen phosphorylase

We have demonstrated the interaction of 125I-labeled calmodulin with glycogen phosphorylase by four techniques: polyacrylamide gel overlay, sucrose density centrifugation, gel filtration chromatography, and affinity chromatography. Phosphorylase b has more affinity for calmodulin than does phosphorylase a. Under all conditions tested, the presence of calmodulin affects neither the enzymatic activity nor any kinetic characteristics of phosphorylase a or b. We present these results as evidence that while binding between calmodulin and phosphorylase clearly exists, it may not have a physiological role.     

Evaluation of novel hyphodermin derivatives as glycogen phosphorylase a inhibitors

The lipophilicity, permeability, solubility, polar surface area and 'rule-of-five' properties were assessed, using QikProp v2.5 (Schr?dinger, Inc.) and ALOGPS 2.1 calculations, for 25 Hyphodermin derivatives. These compounds obeyed the 'rule-of-five', and the calculated physicochemical values were generally within desired limits. All compounds were tested against Glycogen Phosphorylase a (GPa). Four phenyl and benzyl substituted 2-oxo-hexahydro and tetrahydrobenzo[cd]indole carboxylic acids were identified as novel inhibitors of GPa with estimated IC(50) values in the range 0.8-1.3mM. Molecular modelling of these novel inhibitors was used to obtain the main structural features of this class of molecule for future structure-activity relationship studies.     

Protein dynamics of glycogen phosphorylase

The glycogen phosphorylase molecule absorbs the ultraviolet energy of a nitrogen laser to form an excited state of the cofactor. The decay rate of this state has a lifetime of 6.7 microseconds, and its sensitivity to bound substrates presents a new perspective of the mechanism. A careful analysis of the decay curve for native enzyme and cofactor analogues showed that the lifetime depends on the conformation of protein groups at the active site and how the residues change with bound substrate. The reactive ternary complexes obtained from either direction of the reaction yielded the same lifetime, indicating a change in the active-site conformation to a common configuration for the cofactor and substrate phosphate. This configuration indicates an increase in the cofactor 5'-PO4 pKa and a possible proton shuttle. The pyridoxal 5'-pyrophosphate reconstituted enzyme showed no conformational change alone or in the presence of oligosaccharide. This result does not support an electrophilic attack by the 5'-PO4 phosphorus.