Human acylase-1 (hAcyl) is a homodimeric enzyme that catalyzes the hydrolysis of the terminal peptide bond in a protein or the hydrolysis of N-acyl amino acids. The catalytic center of hAcyl has been determined by single-crystal X-ray diffraction with glycine as a cocrystallization aid (Figure 1). D113 H80 E175 Zna Znp E148 m H373 Glycine (crystallization aid) Figure 1 Active site of hAcyl The active site consists of two pentacoordinate Zn ions (A and B, Figure 1) and occurs at the dimer interface. The resting state of the enzyme presumably has a bound water instead of glycine. The proposed mechanism of action by hAcyl involves binding of the substrate, deprotonation of the bound water by a glutamate at position 147 (not shown), and subsequent reaction with the substrate. Addition of water allows release of substrate and return to the resting state. The reacting substrate carbon atom in the mechanism described in the passage undergoes which of the following hybridization state changes during the reaction? (Note: the middle hybridization state refers to an intermediate.) A. sp2 sp3 → sp2 B. sp3 sp2 → sp2 C. sp3 sp? → sp3 D. sp3 sp3 → sp3 Solution: The correct answer is A. This is an Organic Chemistry question that falls under the content category "Structure, function, and reactivity of biologically-relevant molecules." The answer to this question is A. As described in the passage, deprotonation of water is the preliminary step in the reaction sequence. This knowledge and knowledge of the reaction pathways that lead to amide hydrolysis are enough to conclude that the sequence of events involves nucleophilic attack of coordinated hydroxide on the spa-hybridized carbonyl carbon atom to generate an sp3-hybridized and tetrahedral intermediate which subsequently eliminates an amine (after proton transfer) to generate an sp2-hybridized carbon atom. It is a Scientific Reasoning and Problem Solving question because you are asked to bring together theory, evidence, and observations to draw a conclusion.