Bioorthogonal Reactions in Activity-Based Protein Profiling

Activity-based protein profiling (ABPP) is a powerful technique to label and detect active enzyme species within cell lysates, cells, or whole animals. In the last two decades, a wide variety of applications and experimental read-out techniques have been pursued in order to increase our understanding of physiological and pathological processes, to identify novel drug targets, to evaluate selectivity of drugs, and to image probe targets in cells. Bioorthogonal chemistry has substantially contributed to the field of ABPP, as it allows the introduction of tags, which may be bulky or have unfavorable physicochemical properties, at a late stage in the experiment. In this review, we give an overview of the bioorthogonal reactions that have been implemented in ABPP, provide examples of applications of bioorthogonal chemistry in ABPP, and share some thoughts on future directions.

Active-Site Plasticity Is Essential to Carbapenem Hydrolysis by OXA-58 Class D β-Lactamase of Acinetobacter baumannii

ABSTRACTCarbapenem-hydrolyzing class D β-lactamases (CHDLs) are a subgroup of class D β-lactamases, which are enzymes that hydrolyze β-lactams. They have attracted interest due to the emergence of multidrug-resistantAcinetobacter baumannii, which is not responsive to treatment with carbapenems, the usual antibiotics of choice for this bacterium. Unlike other class D β-lactamases, these enzymes efficiently hydrolyze carbapenem antibiotics. To explore the structural requirements for the catalysis of carbapenems by these enzymes, we determined the crystal structure of the OXA-58 CHDL ofA. baumanniifollowing acylation of its active-site serine by a 6α-hydroxymethyl penicillin derivative that is a structural mimetic for a carbapenem. In addition, several point mutation variants of the active site of OXA-58, as identified by the crystal structure analysis, were characterized kinetically. These combined studies confirm the mechanistic relevance of a hydrophobic bridge formed over the active site. This structural feature is suggested to stabilize the hydrolysis-productive acyl-enzyme species formed from the carbapenem substrates of this enzyme. Furthermore, our structural studies provide strong evidence that the hydroxyethyl group of carbapenems samples different orientations in the active sites of CHDLs, and the optimum orientation for catalysis depends on the topology of the active site allowing proper closure of the active site. We propose that CHDLs use the plasticity of the active site to drive the mechanism of carbapenem hydrolysis toward efficiency.

Analysis of an Immoblised Enzyme Reactor with Catalysts Activation

We investigate the behavior of a reaction described by Michaelis-Menten kinetics in an immobilized enzyme reactor (IER). The IER is treated as a well stirred flow reactor, in which the immobilized bounded and unbounded enzyme species are constrained to remain within the reaction vessel. The product species leaves the IER in the reactor outflow. Before the substrate can react with the enzyme, the enzyme must first be activated by absorption of an activator. We use steady state analysis to identify the best operating conditions or the reactor. To this end, we show that the concentration of product is maximized at low residence time whereas the productivity of the reactor is maximized at high residence times.

Immunofluorometric activity-based probe analysis of active KLK6 in biological fluids

Immunoassay measurements of human kallikrein-related peptidases (KLKs) such as prostate-specific antigen (KLK3) are of great value as diagnostic indices of cancer. Despite extensive knowledge of the abundance of immunoreactive KLKs in normal and cancer-related settings, there is little information available about the proportion of immunoreactive KLK that represents active enzyme in such samples. Using KLK6 as a prototype enzyme, we have developed an assay using a serine proteinase-targeted activity-based probe coupled to antibody capture. By employing activity-based labeling, we were able to quantify the proportion of enzymatically active relative to total immunoreactive KLK6 in crude cerebrospinal fluid from routine analyses and ascites fluid from ovarian cancer patients, as well as in supernatants from cancer cell lines. Our approach allowed monitoring of pro-KLK6 conversion to its active enzyme species and demonstrated that up to 5% of immunoreactive KLK6 detected in clinical samples represents active enzyme. We suggest that this new activity-based probe assay will prove of value as a complement to routine KLK immunoassay measurements for validating KLKs as cancer biomarkers.