Paul R. Carey

The glmS ribozyme riboswitch is the first known natural catalytic RNA that employs a small-molecule cofactor. Binding of glucosamine-6-phosphate (GlcN6P) uncovers the latent self-cleavage activity of the RNA, which adopts a catalytically competent conformation that is nonetheless inactive in the absence of GlcN6P. Structural and analogue studies suggest that the amine of GlcN6P functions as a general acid–base catalyst, while its phosphate is important for binding affinity. However, the solution pK_a of the amine is 8.06 ± 0.05, which is not optimal for proton transfer. Here we used Raman crystallography directly to determine the pK_a’s of GlcN6P bound to the glmS ribozyme. Binding to the RNA lowers the pK_a of the amine of GlcN6P to 7.26 ± 0.09 and raises the pK_a of its phosphate to 6.35 ± 0.09. Remarkably, the pK_a’s of these two functional groups are unchanged from their values for free GlcN6P (8.06 ± 0.05 and 5.98 ± 0.05, respectively) when GlcN6P binds to the catalytically inactive but structurally unperturbed G40A mutant of the ribozyme, thus implicating the ribozyme active site guanine in pK_a tuning. This is the first demonstration that a ribozyme can tune the pK_a of a small-molecule ligand. Moreover, the anionic glmS ribozyme in effect stabilizes the neutral amine of GlcN6P by lowering its pK_a. This is unprecedented and illustrates the chemical sophistication of ribozyme active sites.

Extended-spectrum β-lactamases (ESBLs) are derivatives of enzymes such as SHV-1 and TEM-1 that have undergone site-specific mutations that enable them to hydrolyze, and thus inactivate, oxyimino-cephalosporins, such as cefotaxime and ceftazidime. X-ray crystallographic data provide an explanation for this in that the mutations bring about an expansion of the binding pocket by moving a β-strand that forms part of the active site wall. Another characteristic of ESBLs that has remained enigmatic is the fact that they are “hypersusceptible” to inhibition by the mechanism-based inactivators tazobactam, sulbactam, and clavulanic acid. Here, we provide a rationale for this “hypersusceptibility” based on a comparative analysis of the intermediates formed by these compounds with wild-type (WT) SHV-1 β-lactamase and its ESBL variants SHV-2 and SHV-5, which carry the G238S and G238S/E240K substitutions, respectively. A Raman spectroscopic analysis of the reactions in single crystals shows that, compared to WT, the SHV-2 and SHV-5 variants have relatively higher populations of the stable trans-enamine intermediate over the less stable and more easily hydrolyzable cis-enamine and imine co-intermediates. In solution, SHV-2 and SHV-5 also form larger populations of an enamine species compared to SHV-1 as detected by stopped-flow kinetic experiments under single-turnover conditions. Moreover, a simple Raman band shape analysis predicts that the trans-enamine intermediates themselves in SHV-2 and SHV-5 are held in more stable, rigid conformations compared to their trans-enamine analogues in WT SHV-1. As a result of this stabilization, more of the trans-enamine intermediate is formed, which subsequently lowers the K_I values of the mechanism-based inhibitors up to 50-fold in SHV-2 and SHV-5.

Catalytic RNA molecules can achieve rate acceleration by shifting base pK_a values toward neutrality. Prior evidence has suggested that base A38 of the hairpin ribozyme plays an important role in phosphoryl transfer, possibly functioning as a general acid, or by orienting a specific water molecule for proton transfer. To address the role of A38, we used Raman spectroscopy to measure directly the pK_a of the N1-imino moiety in the context of hairpin ribozyme crystals representative of a “precatalytic” conformation. The results revealed that the pK_a of A38 is shifted to 5.46 ± 0.05 relative to 3.68 ± 0.06 derived from a reference solution of the nucleotide AMP. The elevated pK_a correlates well with the first titration point of the macroscopic pH−rate profile of the hairpin ribozyme in solution and strongly supports A38 as a general acid catalyst in bond scission. The results confirm that A38 is protonated before the transition state, which would promote phosphorane development. Overall, the results establish a cogent structure−function paradigm that expands our understanding of how RNA structure can enhance nucleobase reactivity to catalyze biological reactions.

Tazobactam, sulbactam, and clavulanic acid are the only β-lactamase inhibitors in clinical use. Comparative inhibitory activities of clavulanic acid, sulbactam, and tazobactam against clinically important β-lactamases conclude that tazobactam is superior to both clavulanic acid and sulbactam. Thus far, the majority of explanations for this phenomenon have relied on kinetic studies, which report differences in the ligands’ apparent dissociation constants and number of turnovers before inactivation. Due their innate limitations, these investigations do not examine the identity of intermediates on the reaction pathway and relate them to the efficacy of the inhibitors. In the present study, the reactions between the three inhibitors and SHV-1 β-lactamase have been examined in single crystals using a Raman microscope. The results show that tazobactam forms a predominant population of trans-enamine, a chemically inert species, with SHV-1, while clavulanate and sulbactam form a mixture of trans-enamine and two labile species, the cis-enamine and imine. The same reactions are then reexamined using a deacylation-deficient variant, SHV E166A, that has been used to trap acyl-enzyme intermediates for X-ray crystallographic analysis. Our Raman data show that significant differences exist between the wild-type and SHV E166A acyl-enzyme populations. Namely, compared to SHV-1, sulbactam shows significantly smaller populations of cis-enamine and imine in the E166A variant, while clavulanate exists almost exclusively as trans-enamine in the E166A active site. Using clavulanate as an example, we also show that Raman crystallography can provide novel information on the presence of multiple conformers or tautomers for intermediates within a complex reaction pathway. These insights caution against the interpretation of experimental data obtained with deacylation-deficient β-lactamases to make mechanistic conclusions about inhibitors within the enzyme.

The reactions between single crystals of the SHV-1 β-lactamase enzyme and the carbapenems, meropenem, imipenem, and ertapenem, have been studied by Raman microscopy. Aided by quantum mechanical calculations, major populations of two acyl-enzyme species, a labile Δ²-pyrroline and a more tightly bound Δ¹-pyrroline, have been identified for all three compounds. These isomers differ only in the position of the double bond about the carbapenem nucleus. This discovery is consonant with X-ray crystallographic findings that also identified two populations for meropenem bound in SHV-1: one with the acyl C═O group in the oxyanion hole and the second with the acyl group rotated 180° compared to its expected position [Nukaga, M., Bethel, C. R., Thomson, J. M., Hujer, A. M., Distler, A. M., Anderson, V. E., Knox, J. R., and Bonomo, R. A. (2008) J. Am. Chem. Soc. (in press)]. When crystals of the Δ¹- and Δ²-containing acyl-enzymes were exposed to solutions with no carbapenem, rapid deacylation of the Δ² species was observed by kinetic Raman experiments. However, no change in the Δ¹ population was observed over 1 h, the effective lifetime of the crystal. These observations lead to the hypothesis that the stable Δ¹ species is due to the form seen by X-ray with the acyl carbonyl outside the oxyanion hole, while the Δ² species corresponds to the form with the carbonyl inside the oxyanion hole. Soak-in and soak-out Raman experiments also demonstrated that tautomeric exchange between the Δ¹ and Δ² forms does not occur on the crystalline enzyme. When meropenem or ertapenem was reacted with SHV-1 in solution, the Raman difference spectra demonstrated that only a major population corresponding to the Δ¹ acyl-enzyme could be detected. The 1003 cm⁻¹ mode of the phenyl ring positioned on the C3 side chain of ertapenem acts as an effective internal Raman intensity standard, and the ratio of its intensity to that of the 1600 cm⁻¹ feature of Δ¹ provides an estimate of the relative populations of Δ¹. In solution, I₁₆₀₀/I₁₀₀₃ equals 2, and in the crystal, I₁₆₀₀/I₁₀₀₃ equals 1. This is strong evidence that the Δ¹ and Δ² acyl-enzymes in the crystal are present in approximately equal amounts, in agreement with the X-ray data. However, in solution there are twice as many Δ¹ species per Phe group, and this represents approximately 100% of the active sites, which is consistent with the observed inhibition of the enzyme’s activity.

The objective of this study was to determine the molecular factors that lead to β-lactamase inhibitor resistance for the M69V variant in SHV-1 β-lactamase. With mechanism-based inhibitors, the β-lactamase forms an acyl−enzyme intermediate that consists of a trans-enamine derivative in the active site. This study focuses on these intermediates by introducing the E166A mutation that greatly retards deacylation. Thus, by comparing the properties of the E166A and M69V/E166A forms, we can explore the consequences of the resistance mutation at the level of the enamine acyl−enzyme forms. The reactions between the β-lactamase and the inhibitors tazobactam, sulbactam, and clavulanic acid are followed in single crystals of the enzymes by using a Raman microscope. The resulting Raman difference spectroscopic data provide detailed information about conformational events involving the enamine species as well as an estimate of their populations. The Raman difference spectra for each of the inhibitors in the E166A and M69V/E166A variants are very similar. In particular, detailed analysis of the main enamine Raman vibration near 1595 cm^-1 reveals that the structure and flexibility of the enamine fragments are essentially identical for each of the three inhibitors in E166A and in the M69V/E166A double mutant. This finding is in accord with the X-ray-derived structures, presented herein at 1.6−1.75 Å resolution, of the trans-enamine intermediates formed by the three inhibitors in M69V/E166A. However, a comparison of Raman results for M69V/E166A and E166A shows that the M69V mutation results in a 40%, 25%, and negligible reductions in the enamine population when the β-lactamase crystals are soaked in 5 mM tazobactam, clavulanic acid, and sulbactam solutions, respectively. The levels of enamine from tazobactam and clavulanic acid can be increased by increasing the concentrations of inhibitor in the mother liquor. Thus, the sensitivity of population levels to the inhibitor concentration in the mother liquor focuses attention on the properties of the encounter complex preceding acylation. It is proposed that for small ligands, such as tazobactam, sulbactam, and clavulanic acid, the positioning of the lactam ring in the active site in the correct orientation for acylation is only one of a number of poorly defined conformations. For tazobactam and clavulanic acid, the correctly oriented encounter complex is even less likely in the M69V variant, leading to a reduction in the level of inhibition of the enzyme via formation of the acyl−enzyme intermediate and the onset of resistance. Analysis of the X-ray structures of the three intermediates in M69V/E166A demonstrates that, compared to the structures for the E166A form, the oxyanion hole becomes smaller, providing one explanation for why acylation may be less efficient following the M69V substitution.

▪ Abstract Recent studies using a Raman microscope have shown that single protein crystals provide an ideal platform to undertake Raman difference spectroscopic analyses under nonresonance conditions. This approach, termed Raman crystallography, provides a means of characterizing chemical events within the crystal such as ligand binding and enzyme reactions. In many cases Raman crystallography goes hand in hand with X-ray crystallographic studies because the Raman results can inform the X-ray crystallographer about the status of chemical events in the crystal prior to flash freezing and X-ray analysis. In turn, the combined data from the Raman and X-ray analyses are highly synergistic and offer novel perspectives on structure and dynamics in enzyme active sites. In a related area, protein misfolding, Raman microscopy can provide detailed insights into the chemistry of the amyloid plaques associated with Alzheimer's disease and into the intermediates on the α-synuclein protein misfolding pathway implicated in Parkinson's disease.