Decreased Transition-State Analogue Affinity in Isotopically Heavy MTAN with Increased Catalysis.

5'-Methylthioadenosine/S-adenosylhomocysteine nucleosidase from Helicobacter pylori (HpMTAN) demonstrated faster chemistry when expressed as an isotopically heavy protein, with 2H, 13C, and 15N replacing the bulk of normal isotopes. The inverse heavy enzyme isotope effect has been attributed to improved enzyme-reactant interactions causing more frequent transition-state formation ( Proc. Natl. Acad. Sci. U.S.A. 2021, 118, e2109118118). Transition-state analogues stabilize the transient dynamic geometry of the transition state and inform on transition-state dynamics. Here, a slow-onset, tight-binding transition-state analogue of HpMTAN is characterized with heavy and light enzymes. Dissociation constants for the initial encounter complex (Ki) and for the tightly bound complex after slow-onset inhibition (Ki*) with hexylthio-DADMe-Immucillin-A (HTDIA) gave Ki values for light and heavy HpMTAN = 52 ± 10 and 85 ± 13 pM and Ki* values = 5.9 ± 0.3 and 10.0 ± 1.2 pM, respectively. HTDIA dissociates from heavy HpMTAN at 0.063 ± 0.002 min-1, faster than that from light HpMTAN at 0.032 ± 0.004 min-1. These values are consistent with transition-state formation by an improved catalytic site dynamic search and inconsistent with catalytic efficiency proportional to tight binding of the transition state.

Residence Times for Femtomolar and Picomolar Inhibitors of MTANs.

5'-Methylthioadenosine nucleosidases (MTANs) catalyze the hydrolysis of 5'-substituted adenosines to form adenine and 5-substituted ribose. Escherichia coli MTAN (EcMTAN) and Helicobacter pylori MTAN (HpMTAN) form late and early transition states, respectively. Transition state analogues designed for the late transition state bind with fM to pM affinity to both classes of MTANs. Here, we compare the residence times (off-rates) with the equilibrium dissociation constants for HpMTAN and EcMTAN, using five 5'-substituted DADMe-ImmA transition state analogues. The inhibitors dissociate orders of magnitude slower from EcMTAN than from HpMTAN. For example, the slowest release rate was observed for the EcMTAN-HTDIA complex (t1/2 = 56 h), compared to a release rate of t1/2 = 0.3 h for the same complex with HpMTAN, despite similar structures and catalytic sites for these enzymes. Other inhibitors also reveal disconnects between residence times and equilibrium dissociation constants. Residence time is correlated with pharmacological efficacy; thus, experimental analyses of dissociation rates are useful to guide physiological function of tight-binding inhibitors. Steered molecular dynamics simulations for the dissociation of an inhibitor from both EcMTAN and HpMTAN provide atomic level mechanistic insight for the differences in dissociation kinetics and inhibitor residence times for these enzymes.

Inverse heavy enzyme isotope effects in methylthioadenosine nucleosidases.

Heavy enzyme isotope effects occur in proteins substituted with 2H-, 13C-, and 15N-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.

Aminofutalosine Deaminase in the Menaquinone Pathway of Helicobacter pylori.

Helicobacter pylori is a Gram-negative bacterium that is responsible for gastric and duodenal ulcers. H. pylori uses the unusual mqn pathway with aminofutalosine (AFL) as an intermediate for menaquinone biosynthesis. Previous reports indicate that hydrolysis of AFL by 5'-methylthioadenosine nucleosidase (HpMTAN) is the direct path for producing downstream metabolites in the mqn pathway. However, genomic analysis indicates jhp0252 is a candidate for encoding AFL deaminase (AFLDA), an activity for deaminating aminofutolasine. The product, futalosine, is not a known substrate for bacterial MTANs. Recombinant jhp0252 was expressed and characterized as an AFL deaminase (HpAFLDA). Its catalytic specificity includes AFL, 5'-methylthioadenosine, 5'-deoxyadenosine, adenosine, and S-adenosylhomocysteine. The kcat/Km value for AFL is 6.8 × 104 M-1 s-1, 26-fold greater than that for adenosine. 5'-Methylthiocoformycin (MTCF) is a slow-onset inhibitor for HpAFLDA and demonstrated inhibitory effects on H. pylori growth. Supplementation with futalosine partially restored H. pylori growth under MTCF treatment, suggesting AFL deamination is significant for cell growth. The crystal structures of apo-HpAFLDA and with MTCF at the catalytic sites show a catalytic site Zn2+ or Fe2+ as the water-activating group. With bound MTCF, the metal ion is 2.0 Å from the sp3 hydroxyl group of the transition state analogue. Metabolomics analysis revealed that HpAFLDA has intracellular activity and is inhibited by MTCF. The mqn pathway in H. pylori bifurcates at aminofutalosine with HpMTAN producing adenine and depurinated futalosine and HpAFLDA producing futalosine. Inhibition of cellular HpMTAN or HpAFLDA decreased the cellular content of menaquinone-6, supporting roles for both enzymes in the pathway.