Protein lysine methyltransferases (PKMTs) catalyze the methylation of protein substrates, and their dysregulation has been linked to many diseases, including cancer. Accumulated evidence suggests that the reaction path of PKMT-catalyzed methylation consists of the formation of a cofactor(cosubstrate)-PKMT-substrate complex, lysine deprotonation through dynamic water channels, and a nucleophilic substitution (S N 2) transition state for transmethylation. However, the molecular characters of the proposed process remain to be elucidated experimentally. Here we developed a matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF-MS) method and corresponding mathematic matrix to determine precisely the ratios of isotopically methylated peptides. This approach may be generally applicable for examining the kinetic isotope effects (KIEs) of posttranslational modifying enzymes. Protein lysine methyltransferase SET8 is the sole PKMT to monomethylate histone 4 lysine 20 (H4K20) and its function has been implicated in normal cell cycle progression and cancer metastasis. We therefore implemented the MSbased method to measure KIEs and binding isotope effects (BIEs) of the cofactor S-adenosyl-L-methionine (SAM) for SET8-catalyzed H4K20 monomethylation. A primary intrinsic 13 C KIE of 1.04, an inverse intrinsic α-secondary CD 3 KIE of 0.90, and a small but statistically significant inverse CD 3 BIE of 0.96, in combination with computational modeling, revealed that SET8-catalyzed methylation proceeds through an early, asymmetrical S N 2 transition state with the C-N and C-S distances of 2.35-2.40 Å and 2.00-2.05 Å, respectively. This transition state is further supported by the KIEs, BIEs, and steadystate kinetics with the SAM analog Se-adenosyl-L-selenomethionine (SeAM) as a cofactor surrogate. The distinct transition states between protein methyltransferases present the opportunity to design selective transition-state analog inhibitors.S tepwise progression of an enzyme-catalyzed chemical reaction is accompanied by changes of bond orders and vibrational modes involved with specific atoms of the reactant(s) (1, 2). Such changes can be traced experimentally by measuring the ratios of turnover rates [kinetic isotope effects (KIEs)] or binding affinities [binding isotope effects (BIEs)] of the reactant(s) when the relevant atoms are replaced by heavy isotopes (3, 4). KIEs and BIEs are thus useful parameters for elucidating transition-state (TS) structures and catalytic mechanisms, which sometimes cannot be elucidated readily through sole measurement of steady-state kinetics (5-9). A sufficient set of KIEs and BIEs at the positions involved with bond motions can afford electrostatic and geometric constraints, when combined with computational modeling, to define an enzymatic TS (10-12). This information provides not only the atomic resolution of the transient structure at the highest energy summit along the reaction path, but also structural guidance for designing tight-binding TS analog inhibitors (13...

Purpose Prolonged ureteroscopy (URS) is associated with complications including ureteral perforation, stricture, and urosepsis. As laser lithotripsy is one of the most common urologic procedures, small cost savings per case can have a large financial impact. This retrospective study was designed to determine if Thulium fiber laser (TFL) lithotripsy decreases operative time and costs compared to standard Holmium:YAG (Ho:YAG) lithotripsy without pulse modulation. Methods A retrospective review of URS with laser lithotripsy was conducted for 152 cases performed from August 2020 to January 2021. Variables including cumulative stone size, location, chemical composition, prior ureteral stenting, and ureteral access sheath use were recorded for each case. A cost benefit analysis was performed to show projected cost savings due to potentially decreased operative times. Results Compared to Ho:YAG, use of TFL resulted in an average decrease of 12.9 min per case ( p = .021, 95% CI [2.03–23.85]). In subgroup analysis of cases with cumulative stone diameter less than 15 mm, the difference was 14.0 min ( p = .007, CI [3.95–23.95]). For cases less than 10 mm, the mean difference was 17.3 min in favor of TFL ( p = .002, 95% CI [6.89–27.62]). This ~ 13 min reduction in operative time resulted in saving $440/case in direct operating room costs giving our institution a range of $294,000 to $381,900 savings per year. Conclusions TFL has a significantly shorter operative time and decreased cost when compared to the standard Ho:YAG for equivalent kidney stone and patient characteristics. Longer term follow up is needed to see if recurrence rates are affected.

Regulation of the enzootic cycle in Borrelia burgdorferi requires a shift to the RNA polymerase alternative sigma factor, RpoS. We used in vitro and in vivo assays to assess the relative importance of the putative Shine-Dalgarno sequence and its sequestration for the translational efficiency of rpoS. We created mutant leader regions in which we either removed the Shine-Dalgarno sequence, disrupted the secondary structure or both. Binding assays and toeprint assays demonstrated that both the presence and the availability of the Shine-Dalgarno sequence are important to the efficiency and specificity of ribosome binding. Adding a DsrABb mimic in the form of a single-stranded DNA oligonucleotide increased the level and specificity of binding of RL, presumably by making the Shine-Dalgarno sequence available for binding. In in vivo assays we confirmed that the Shine-Dalgarno sequence must be both present and un-sequestered in order for translation to proceed efficiently. The RL transcript was significantly better translated in B. burgdorferi at 37°C than at 26°C, lending support to the hypothesis that DsrABb acts as a temperature-dependent stimulator of translation. These studies demonstrate that translational regulation of gene expression in B. burgdorferi may be an important mechanism for responding to environmental signals important in the enzootic cycle.

Transition state stabilization is essential for rate acceleration of enzymatic reactions. Despite extensive studies on various transition state structures of enzymes, an intriguing puzzle is whether an enzyme can accommodate multiple transition states (TS) to catalyze a chemical reaction. It is experimentally challenging to interrogate this proposition in terms of the choices of suitable enzymes and the feasibility to distinguish multiple TS. As a paradigm with the protein lysine methyltransferase (PKMT) SET7/9 paired with its physiological substrates H3 and p53, their TS were solved with experimental kinetic isotope effects as computational constraints. Remarkably, SET7/9 adopts two structurally distinct TS---a nearly symmetric S N 2 and an extremely early S N 2---for H3K4 and p53K372 methylation, respectively. The two TS are also different from those previously revealed for other PKMTs. The setting of multiple TS is expected to be essential for SET7/9 and likely other PKMTs to act on broad substrates with high efficiency.