Transition-state structures for N-glycoside hydrolysis of AMP by acid and by AMP nucleosidase in the presence and absence of allosteric activator

Abstract

The mechanism of acid and enzymatic hydrolysis of the N-glycosidic bond of AMP has been investigated by fitting experimentally observed kinetic isotope effects [Parkin, D. W., & Schramm, V. L. (1987) Biochemistry (preceding paper in this issue)] to calculated kinetic isotope effects for proposed transition-state structures. The sensitivity of the transition-state calculations was tested by "arying the transition-state structure and comparing changes in the calculated kinetic isotope effects with the experimental values of the isotope effect measurements. The kinetic isotope effects for the acid-catalyzed hydrolysis of AMP are best explained by a transition state with considerable oxycarbonium character in the ribose ring, significant bonding remaining to the departing adenine ring, participation of a water nucleophile, and protonation of the adenine ring. A transition-state structure without preassociation of the water nucleophile cannot be eliminated by the data. Enzymatic hydrolysis of the N-glycosidic bond of AMP by AMP nucleosidase from Azotobacter vinelandii was analyzed in the absence and presence of MgATP, the allosteric activator that increases Vmax approximately 200-fold. The transition states for enzyme-catalyzed hydrolysis that best explain the kinetic isotope effects involve early SN1 transition states with significant bond order in the glycosidic bond and protonation of the adenine base. The enzyme enforces participation of an enzyme-bound water molecule, which has weak bonding to C1' in the transition state. Activation of AMP nucleosidase by MgATP causes the bond order of the glycosidic bond in the transition state to increase significantly. Hyperconjugation in the ribosyl group is altered by enzymatic stabilization of the oxycarbonium ion. This change is consistent with the interaction of an amino acid on the enzyme. Together, these changes stabilize a carboxonium-like transition-state complex that occurs earlier in the reaction pathway than in the absence of allosteric activator. In addition to the allosteric changes that alter transition-state structure, the presence of other inductive effects that are unobserved by kinetic isotope measurements is also likely to increase the catalytic rate.

Structure and Inhibition of a Quorum Sensing Target from Streptococcus pneumoniae

“…This relationship predicts that transition state analogues based on the SAH scaffold would prefer the E. coli enzyme by a smaller difference that those based on the MTA structure. Homocys-DADMe-ImmA [22] is a 610 pM inhibitor of S. pneumoniae MTAN and a 6 pM inhibitor of E. coli MTAN (29), in good agreement with the k cat /K m values for these enzymes and substrates. We are not aware of other examples that demonstrate this close relationship between k cat /K m and transition state analogue binding with closely related isozymes and different substrates.…”

“…DADMeImmucillins were also modified by incorporating hydrophobic groups to replace the 5′-methyl group and many of the 5′-hydrophobic DADMe-ImmA analogues are slow-onset tight binding inhibitors ( Figure 5 S. pneumoniae MTAN has dual substrate specificity for MTA [19] and SAH [20] and gives a K m for SAH of 13 μM, almost 2 fold lower than for MTA. This difference is also seen in the transition state analogues, where homocysteinyl-DADMe-ImmA [22] is a slow onset inhibitor with a K i * of 610 pM, 18-fold increased affinity relative to MT-DADMe-ImmA [34]. Adenosine and methylthioadenosine are not substrates, and accordingly, Immucillin-A [17] is a poor inhibitor with a K i of 30 μM and MT-Immucillin-H [16] or Immucillin-H [18] are not inhibitors at micromolar concentrations.…”

“…Adenine is recycled to the adenine nucleotide pool by adenine phosphoribosyltransferase (18) and 5-methylthio-D-ribose is converted into methionine (19). Disruption of MTAN catalytic activity is therefore expected to affect adenine and methionine salvage, polyamine biosynthesis and quorum sensing.Transition states of nucleoside and nucleotide N-ribosyl hydrolases involve formation of ribosyl oxacarbenium ions, where the N-ribosidic bond is cleaved to variable extents without significant bond order to the attacking water nucleophile (20)(21)(22). The oxacarbenium ion transition state structures of N-ribosyl transferases can be grouped into early transition states that have significant bond order to the leaving group and late transition states with little or no bond order to the leaving group.…”

Enzymatic Transition States and Transition State Analog Design

“…This provides the opportunity to determine whether allosteric activation is capable of changing the chemical nature of the transition state or only serves to change energies of activation (altered rate constants) for steps through the reaction cycle. Systematic determination of kinetic isotope effects is made possible by the combined chemical and enzymatic synthesis of AMP ( Figure 6) with the isotopic labels indicated in Figure 7 (54)(55)(56).…”

Influence of Allosteric Regulators on Individual Steps in the Reaction Catalyzed by Mycobacterium tuberculosis 2-Hydroxy-3-oxoadipate Synthase

“…In addition, the presence of allosteric activators could change the nature of the transition state as in AMP nucleosidase (44,45). Based on the dependence of K i of MSP and K d of MSP-ThDP adduct on the presence of AcCoA, we suggest that accounting for allosteric regulators could aid in developing mechanism-based inhibitors with improved potency and enhanced specificity against homologs that use different mechanisms of allostery.…”