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Heavy enzyme isotope effects occur in proteins substituted with 2 H-, 13 C-, and 15 N-enriched amino acids. Mass alterations perturb femtosecond protein motions and have been used to study the linkage between fast motions and transition-state barrier crossing. Heavy enzymes typically show slower rates for their chemical steps. Heavy bacterial methylthioadenosine nucleosidases (MTANs from Helicobactor pylori and Escherichia coli ) gave normal isotope effects in steady-state kinetics, with slower rates for the heavy enzymes. However, both enzymes revealed rare inverse isotope effects on their chemical steps, with faster chemical steps in the heavy enzymes. Computational transition-path sampling studies of H. pylori and E. coli MTANs indicated closer enzyme-reactant interactions in the heavy MTANs at times near the transition state, resulting in an improved reaction coordinate geometry. Specific catalytic interactions more favorable for heavy MTANs include improved contacts to the catalytic water nucleophile and to the adenine leaving group. Heavy bacterial MTANs depart from other heavy enzymes as slowed vibrational modes from the heavy isotope substitution caused improved barrier-crossing efficiency. Improved sampling frequency and reactant coordinate distances are highlighted as key factors in MTAN transition-state stabilization., Competing Interests: The authors declare no competing interest.

Transition path-sampling calculations with several enzymes have indicated that local catalytic site femtosecond motions are linked to transition state barrier crossing. Experimentally, femtosecond motions can be perturbed by labeling the protein with amino acids containing 13 C, 15 N, and nonexchangeable 2 H. A slowed chemical step at the catalytic site with variable effects on steady-state kinetics is usually observed for heavy enzymes. Heavy human purine nucleoside phosphorylase (PNP) is slowed significantly ( k chem light / k chem heavy = 1.36). An asparagine (Asn243) at the catalytic site is involved in purine leaving-group activation in the PNP catalytic mechanism. In a PNP produced with isotopically heavy asparagines, the chemical step is faster ( k chem light / k chem heavy = 0.78). When all amino acids in PNP are heavy except for the asparagines, the chemical step is also faster ( k chem light / k chem heavy = 0.71). Substrate-trapping experiments provided independent confirmation of improved catalysis in these constructs. Transition path-sampling analysis of these partially labeled PNPs indicate altered femtosecond catalytic site motions with improved Asn243 interactions to the purine leaving group. Altered transition state barrier recrossing has been proposed as an explanation for heavy-PNP isotope effects but is incompatible with these isotope effects. Rate-limiting product release governs steady-state kinetics in this enzyme, and kinetic constants were unaffected in the labeled PNPs. The study suggests that mass-constrained femtosecond motions at the catalytic site of PNP can improve transition state barrier crossing by more frequent sampling of essential catalytic site contacts., Competing Interests: The authors declare no conflict of interest.

Plasmodium falciparum causes the most lethal form of human malaria and is a global health concern. The parasite responds to antimalarial therapies by developing drug resistance. The continuous development of new antimalarials with novel mechanisms of action is a priority for drug combination therapies. The use of transition-state analog inhibitors to block essential steps in purine salvage has been proposed as a new antimalarial approach. Mutations that reduce transition-state analog binding are also expected to reduce the essential catalytic function of the target. We have previously reported that inhibition of host and P. falciparum purine nucleoside phosphorylase ( Pf PNP) by DADMe-Immucillin-G (DADMe-ImmG) causes purine starvation and parasite death in vitro and in primate infection models. P. falciparum cultured under incremental DADMe-ImmG drug pressure initially exhibited increased Pf PNP gene copy number and protein expression. At increased drug pressure, additional Pf PNP gene copies appeared with point mutations at catalytic site residues involved in drug binding. Mutant Pf PNPs from resistant clones demonstrated reduced affinity for DADMe-ImmG, but also reduced catalytic efficiency. The catalytic defects were partially overcome by gene amplification in the region expressing Pf PNP. Crystal structures of native and mutated Pf PNPs demonstrate altered catalytic site contacts to DADMe-ImmG. Both point mutations and gene amplification are required to overcome purine starvation induced by DADMe-ImmG. Resistance developed slowly, over 136 generations (2 136 clonal selection). Transition-state analog inhibitors against Pf PNP are slow to induce resistance and may have promise in malaria therapy., Competing Interests: The authors declare no conflict of interest.

Heavy-enzyme isotope effects ( 15 N-, 13 C-, and 2 H-labeled protein) explore mass-dependent vibrational modes linked to catalysis. Transition path-sampling (TPS) calculations have predicted femtosecond dynamic coupling at the catalytic site of human purine nucleoside phosphorylase (PNP). Coupling is observed in heavy PNPs, where slowed barrier crossing caused a normal heavy-enzyme isotope effect ( k chem light / k chem heavy > 1.0). We used TPS to design mutant F159Y PNP, predicted to improve barrier crossing for heavy F159Y PNP, an attempt to generate a rare inverse heavy-enzyme isotope effect ( k chem light / k chem heavy < 1.0). Steady-state kinetic comparison of light and heavy native PNPs to light and heavy F159Y PNPs revealed similar kinetic properties. Pre-steady-state chemistry was slowed 32-fold in F159Y PNP. Pre-steady-state chemistry compared heavy and light native and F159Y PNPs and found a normal heavy-enzyme isotope effect of 1.31 for native PNP and an inverse effect of 0.75 for F159Y PNP. Increased isotopic mass in F159Y PNP causes more efficient transition state formation. Independent validation of the inverse isotope effect for heavy F159Y PNP came from commitment to catalysis experiments. Most heavy enzymes demonstrate normal heavy-enzyme isotope effects, and F159Y PNP is a rare example of an inverse effect. Crystal structures and TPS dynamics of native and F159Y PNPs explore the catalytic-site geometry associated with these catalytic changes. Experimental validation of TPS predictions for barrier crossing establishes the connection of rapid protein dynamics and vibrational coupling to enzymatic transition state passage., Competing Interests: The authors declare no conflict of interest.

Human DNA methyltransferase 1 (DNMT1) maintains the epigenetic state of DNA by replicating CpG methylation signatures from parent to daughter strands, producing heritable methylation patterns through cell divisions. The proposed catalytic mechanism of DNMT1 involves nucleophilic attack of Cys(1226) to cytosine (Cyt) C6, methyl transfer from S-adenosyl-l-methionine (SAM) to Cyt C5, and proton abstraction from C5 to form methylated CpG in DNA. Here, we report the subangstrom geometric and electrostatic structure of the major transition state (TS) of the reaction catalyzed by human DNMT1. Experimental kinetic isotope effects were used to guide quantum mechanical calculations to solve the TS structure. Methyl transfer occurs after Cys(1226) attack to Cyt C6, and the methyl transfer step is chemically rate-limiting for DNMT1. Electrostatic potential maps were compared for the TS and ground states, providing the electronic basis for interactions between the protein and reactants at the TS. Understanding the TS of DNMT1 demonstrates the possibility of using similar analysis to gain subangstrom geometric insight into the complex reactions of epigenetic modifications.

Nuclear receptor SET domain containing protein 2 (NSD2) catalyzes the methylation of histone H3 lysine 36 (H3K36). It is a determinant in Wolf-Hirschhorn syndrome and is overexpressed in human multiple myeloma. Despite the relevance of NSD2 to cancer, there are no potent, selective inhibitors of this enzyme reported. Here, a combination of kinetic isotope effect measurements and quantum chemical modeling was used to provide subangstrom details of the transition state structure for NSD2 enzymatic activity. Kinetic isotope effects were measured for the methylation of isolated HeLa cell nucleosomes by NSD2. NSD2 preferentially catalyzes the dimethylation of H3K36 along with a reduced preference for H3K36 monomethylation. Primary Me-(14)C and (36)S and secondary Me-(3)H3, Me-(2)H3, 5'-(14)C, and 5'-(3)H2 kinetic isotope effects were measured for the methylation of H3K36 using specifically labeled S-adenosyl-l-methionine. The intrinsic kinetic isotope effects were used as boundary constraints for quantum mechanical calculations for the NSD2 transition state. The experimental and calculated kinetic isotope effects are consistent with an SN2 chemical mechanism with methyl transfer as the first irreversible chemical step in the reaction mechanism. The transition state is a late, asymmetric nucleophilic displacement with bond separation from the leaving group at (2.53 Å) and bond making to the attacking nucleophile (2.10 Å) advanced at the transition state. The transition state structure can be represented in a molecular electrostatic potential map to guide the design of inhibitors that mimic the transition state geometry and charge.

Computational chemistry predicts that atomic motions on the femtosecond timescale are coupled to transition-state formation (barrier-crossing) in human purine nucleoside phosphorylase (PNP). The prediction is experimentally supported by slowed catalytic site chemistry in isotopically labeled PNP (13C, 15N, and 2H). However, other explanations are possible, including altered volume or bond polarization from carbon-deuterium bonds or propagation of the femtosecond bond motions into slower (nanoseconds to milliseconds) motions of the larger protein architecture to alter catalytic site chemistry. We address these possibilities by analysis of chemistry rates in isotope-specific labeled PNPs. Catalytic site chemistry was slowed for both [2H]PNP and [13C, 15N]PNP in proportion to their altered protein masses. Secondary effects emanating from carbon-deuterium bond properties can therefore be eliminated. Heavy-enzyme mass effects were probed for local or global contributions to catalytic site chemistry by generating [15N, 2H]His8-PNP. Of the eight His per subunit, three participate in contacts to the bound reactants and five are remote from the catalytic sites. [15N, 2H]His8-PNP had reduced catalytic site chemistry larger than proportional to the enzymatic mass difference. Altered barrier crossing when only His are heavy supports local catalytic site femtosecond perturbations coupled to transition-state formation. Isotope-specific and amino acid specific labels extend the use of heavy enzyme methods to distinguish global from local isotope effects.

Transition state analogs mimic the geometry and electronics of the transition state of enzymatic reactions. These molecules bind to the active site of the enzyme much tighter than substrate and are powerful noncovalent inhibitors. Immucillin-H (ImmH) and 4'-deaza-1'-aza-2'-deoxy-9-methylene Immucillin-H (DADMe-ImmH) are picomolar inhibitors of human purine nucleoside phosphorylase (hPNP). Although both molecules are electronically similar to the oxocarbenium-like dissociative hPNP transition state, DADMe-ImmH is more potent than ImmH. DADMe-ImmH captures more of the transition state binding energy by virtue of being a closer geometric match to the hPNP transition state than ImmH. A consequence of these similarities is that the active site of hPNP exerts greater distortional forces on ImmH than on DADMe-ImmH to "achieve" the hPNP transition state geometry. By using magic angle spinning solid-state NMR to investigate stable isotope-labeled ImmH and DADMe-ImmH, we have explored the difference in distortional binding of these two inhibitors to hPNP. High-precision determinations of internuclear distances from NMR recoupling techniques, rotational echo double resonance, and rotational resonance, have provided unprecedented atomistic insight into the geometric changes that occur upon binding of transition state analogs. We conclude that hPNP stabilizes conformations of these chemically distinct analogs having distances between the cation and leaving groups resembling those of the known transition state.

HIV-1 protease is an important target for the treatment of HIV/AIDS. However, drug resistance is a persistent problem and new inhibitors are needed. An approach toward understanding enzyme chemistry, the basis of drug resistance, and the design of powerful inhibitors is to establish the structure of enzymatic transition states. Enzymatic transition structures can be established by matching experimental kinetic isotope effects (KIEs) with theoretical predictions. However, the HIV-1 protease transition state has not been previously resolved using these methods. We have measured primary (14)C and (15)N KIEs and secondary (3)H and (18)O KIEs for native and multidrug-resistant HIV-1 protease (I84V). We observed (14)C KIEs ((14)V/K) of 1.029 ± 0.003 and 1.025 ± 0.005, (15)N KIEs ((15)V/K) of 0.987 ± 0.004 and 0.989 ± 0.003, (18)O KIEs ((18)V/K) of 0.999 ± 0.003 and 0.993 ± 0.003, and (3)H KIEs ((3)V/K) KIEs of 0.968 ± 0.001 and 0.976 ± 0.001 for the native and I84V enzyme, respectively. The chemical reaction involves nucleophilic water attack at the carbonyl carbon, proton transfer to the amide nitrogen leaving group, and C-N bond cleavage. A transition structure consistent with the KIE values involves proton transfer from the active site Asp-125 (1.32 Å) with partial hydrogen bond formation to the accepting nitrogen (1.20 Å) and partial bond loss from the carbonyl carbon to the amide leaving group (1.52 Å). The KIEs measured for the native and I84V enzyme indicate nearly identical transition states, implying that a true transition-state analogue should be effective against both enzymes.

Contributions of fast (femtosecond) dynamic motion to barrier crossing at enzyme catalytic sites is in dispute. Human purine nucleoside phosphorylase (PNP) forms a ribocation-like transition state in the phosphorolysis of purine nucleosides and fast protein motions have been proposed to participate in barrier crossing. In the present study, (13)C-, (15)N-, (2)H-labeled human PNP (heavy PNP) was expressed, purified to homogeneity, and shown to exhibit a 9.9% increase in molecular mass relative to its unlabeled counterpart (light PNP). Kinetic isotope effects and steady-state kinetic parameters were indistinguishable for both enzymes, indicating that transition-state structure, equilibrium binding steps, and the rate of product release were not affected by increased protein mass. Single-turnover rate constants were slowed for heavy PNP, demonstrating reduced probability of chemical barrier crossing from enzyme-bound substrates to enzyme-bound products. In a second, independent method to probe barrier crossing, heavy PNP exhibited decreased forward commitment factors, also revealing mass-dependent decreased probability for barrier crossing. Increased atomic mass in human PNP alters bond vibrational modes on the femtosecond time scale and reduces on-enzyme chemical barrier crossing. This study demonstrates coupling of enzymatic bond vibrations on the femtosecond time scale to barrier crossing.

Inhibition of human purine nucleoside phosphorylase (PNP) stops growth of activated T-cells and the formation of 6-oxypurine bases, making it a target for leukemia, autoimmune disorders, and gout. Four generations of ribocation transition-state mimics bound to PNP are structurally characterized. Immucillin-H (K*i(1/4) 58 pM, first generation)contains an iminoribitol cation with four asymmetric carbons. DADMe-Immucillin-H (K*i(1/4) 9 pM, second-generation),uses a methylene-bridged dihydroxypyrrolidine cation with twoasymmetric centers.DATMe-Immucillin-H (K*i(1/4)9 pM, third-generation) contains an open-chain amino alcohol cation with two asymmetric carbons. SerMe-ImmH (K*i(1/4) 5 pM, fourth-generation) uses achiral dihydroxyaminoalcohol seramide as the ribocation mimic. Crystal structures of PNPs establish features of tight binding to be; 1) ion-pair formation between bound phosphate (or its mimic) and inhibitor cation, 2) leaving-group interactions to N1, O6, and N7 of 9-deazahypoxanthine, 3) interaction between phosphate and inhibitor hydroxyl groups, and 4) His257 interacting with the 5'-hydroxyl group. The first generation analogue is an imperfect fit to the catalytic site with a long ion pair distance between the iminoribitol and bound phosphate and weaker interactions to the leaving group. Increasing the ribocation to leaving-group distance in the second- to fourth-generation analogues provides powerful binding interactions and a facile synthetic route to powerful inhibitors. Despite chemical diversity in the four generations of transition-state analogues, the catalytic site geometry is almost the same for all analogues. Multiple solutions in transition-state analogue design are available to convert the energy of catalytic rate enhancement to binding energy in human PNP.

Ricin A-chain (RTA) and saporin-L1 (SAP) catalyze adenosine depurination of 28S rRNA to inhibit protein synthesis and cause cell death. We present the crystal structures of RTA and SAP in complex with transition state analogue inhibitors. These tight-binding inhibitors mimic the sarcin-ricin recognition loop of 28S rRNA and the dissociative ribocation transition state established for RTA catalysis. RTA and SAP share unique purine-binding geometry with quadruple pi-stacking interactions between adjacent adenine and guanine bases and 2 conserved tyrosines. An arginine at one end of the pi-stack provides cationic polarization and enhanced leaving group ability to the susceptible adenine. Common features of these ribosome-inactivating proteins include adenine leaving group activation, a remarkable lack of ribocation stabilization, and conserved glutamates as general bases for activation of the H(2)O nucleophile. Catalytic forces originate primarily from leaving group activation evident in both RTA and SAP in complex with transition state analogues.

Nicotinamide phosphoribosyltransferase (NAMPT) is highly evolved to capture nicotinamide (NAM) and replenish the nicotinamide adenine dinucleotide (NAD(+)) pool during ADP-ribosylation and transferase reactions. ATP-phosphorylation of an active-site histidine causes catalytic activation, increasing NAM affinity by 160,000. Crystal structures of NAMPT with catalytic site ligands identify the phosphorylation site, establish its role in catalysis, demonstrate unique overlapping ATP and phosphoribosyltransferase sites, and establish reaction coordinate motion. NAMPT structures with beryllium fluoride indicate a covalent H247-BeF(3)(-) as the phosphohistidine mimic. Activation of NAMPT by H247-phosphorylation causes stabilization of the enzyme-phosphoribosylpyrophosphate complex, permitting efficient capture of NAM. Reactant and product structures establish reaction coordinate motion for NAMPT to be migration of the ribosyl anomeric carbon from the pyrophosphate leaving group to the nicotinamide-N1 while the 5-phosphoryl group, the pyrophosphate moiety, and the nicotinamide ring remain fixed in the catalytic site.

Transition path sampling (TPS) has been applied to the chemical step of human purine nucleoside phosphorylase (PNP). The transition path ensemble provides insight into the detailed mechanistic dynamics and atomic motion involved in transition state passage. The reaction mechanism involves early loss of the ribosidic bond to form a transition state with substantial ribooxacarbenium ion character, followed by dynamic motion from the enzyme and a conformational change in the ribosyl group leading to migration of the anomeric carbon toward phosphate, to form the product ribose 1-phosphate. Calculations of the commitment probability along reactive paths demonstrated the presence of a broad energy barrier at the transition state. TPS identified (i) compression of the O4'...O5' vibrational motion, (ii) optimized leaving group interactions, and (iii) activation of the phosphate nucleophile as the reaction proceeds through the transition state region. Dynamic motions on the femtosecond timescale provide the simultaneous optimization of these effects and coincide with transition state formation.

Transition-state theory has led to the design of Immucillin-H (Imm-H), a picomolar inhibitor of purine nucleoside phosphorylase (PNP). In humans, PNP is the only route for degradation of deoxyguanosine, and genetic deficiency of this enzyme leads to profound T cell-mediated immunosuppression. This study reports the biological effects and mechanism of action of Imm-H on malignant T cell lines and on normal activated human peripheral T cells. Imm-H inhibits the growth of malignant T cell leukemia lines with the induction of apoptosis. Imm-H also inhibits activated normal human T cells after antigenic stimulation in vitro. However, Imm-H did not inhibit malignant B cells, colon cancer cell lines, or normal human nonstimulated T cells, demonstrating the selective activity of Imm-H. The effects on leukemia cells were mediated by the cellular phosphorylation of deoxyguanosine and the accumulation of dGTP, an inhibitor of ribonucleotide diphosphate reductase. Cells were protected from the toxic effects of Imm-H when deoxyguanosine was absent or when deoxycytidine was present. Guanosine incorporation into nucleic acids was selectively blocked by Imm-H with no effect on guanine, adenine, adenosine, or deoxycytidine incorporation. Imm-H may have clinical potential for treatment of human T cell leukemia and lymphoma and for other diseases characterized by abnormal activation of T lymphocytes. The design of Imm-H from an enzymatic transition-state analysis exemplifies a powerful approach for developing high-affinity enzyme inhibitors with pharmacologic activity.

The divalent cation-binding properties of the human insulin receptor tyrosine kinase domain were examined kinetically and by electron paramagnetic resonance and circular dichroic spectroscopy. The protein-tyrosine kinase activity of the purified cytoplasmic domain can be activated nearly 10-fold by 3 mM Mn2+ in the presence or absence of 5 mM Mg2+. Electron paramagnetic resonance spectra of the purified, acid-denatured kinase domain and assays of EDTA-treated kinase show that the purified protein does not possess residual, tightly bound Mn2+. Electron paramagnetic resonance spectroscopy was used to directly measure the binding constant of the kinase domain for Mn2+. The results indicate that the recombinant cytoplasmic domain of the human insulin receptor does not bind Mn2+ tightly in the absence or presence of MgATP (Kd greater than 0.8 mM). Furthermore, the enzyme does not show a strong preference for MnATP binding when both MgATP and MnATP are present. The far-ultraviolet circular dichroic spectrum of this domain is characterized by a negative maximum at 207 nm. In the presence of Mn2+, but not Mg2+, changes in the mean residue-weight ellipticity at 207 nm occur that are consistent with a decrease in alpha-helical content. The addition of ATP to Mn2(+)-bound protein does not further perturb the spectrum. We conclude that Mn2+ ions, although they bind weakly, induce an activating conformational change in the secondary structure of the human insulin receptor cytoplasmic domain. Activation by Mn2+ is unlikely to be significant in intact cells, but it may mimic the action of a physiological activator.

Purified beta-1,4-N-acetylmuramoylhydrolase (muramidase-1; EC 3.2.1.17) of Streptococcus faecium ATCC 9790 has been shown to be covalently substituted with approximately 12 mol equivalents of monomeric 5-mercaptouridine monophosphate. All 12 residues are present on the proteolytically processed 87-kDa active form of the enzyme. A peptide fragment containing 5-mercaptouridine, tyrosine, alanine, glycine, and leucine was isolated consistent with an O-phosphate linkage of the nucleotide to tyrosine.