Typical biochemistry labs exploring basic enzyme activity rely on costly, time-consuming protein purification and rarely explore enzyme function in situ. Further, complex purification procedures leave little room for novelty in experimental design. Here we present an inquiry-driven laboratory exercise for biochemistry undergraduates and adaptations for a general education science course. Each student designs a unique experiment to test their hypothesis regarding the nature of avocado browning in a three-hour span. In the presence of oxygen, polyphenol oxidases (PPO) catalyze oxidation of phenolic compounds into quinones, the polymerization of which creates the visible browning of many cut fruits. Avocado fruit, a source of both enzyme and substrate, is a safe, low-cost vehicle for semi-quantitative experimentation. During the incubation, biochemistry students use the Protein Data Bank and primary literature to understand the structure-function relationship of PPO and other molecular components of the avocado. Non-major students discuss how pH, temperature, and substrate availability affect PPO. Visible browning pigments appear on a controllable time scale. Students can photograph results to create a figure to accompany semi-quantitative analysis of experimental results in a single lab period. Since avocados are familiar foods and select test reagents are generally recognized as safe, the optimal protocol investigated in the lab can be further applied to best practices in the kitchen in everyday life, promoting the transfer of knowledge learned in the classroom to practical environments.

Enzyme rates and kinetics are key components used by biochemists to understand how enzymes function. However, students often find it difficult to understand how these experiments are performed and how they reflect enzyme behavior in solution. The microscopic behaviors which compose K_M, V_max, and other kinetic parameters are not easy to see, hindering clear incorporation of kinetics into students' biochemical knowledge. We describe a set of in-class activities where students act as enzymes in order to clarify the behavior of enzymes in solution and to develop a more robust understanding of how kinetics describe this behavior. In the first demonstration, students observe how the rate of candy unwrapping changes over time in a closed system showing how products can slow the progress of an enzyme reaction. In the second demonstration, students observe how substrate concentration and the rate of enzyme reactions are linked and eventually saturate. A final aspect of this lesson helps students learn how to fit their own data to calculate the kinetic values V_max and K_M. Extensions of this activity to enzyme inhibition and active site structure are also described. Students felt more confident in their understanding of enzyme kinetics and action after performing these activities.

Enzyme kinetics and the impacts of inhibitors on the enzyme's maximal velocity and ability to bind substrates are important topics in cell biology and biochemistry. However, these topics can be difficult for students to grasp when instructed using a traditional lecture format. Teaching biological concepts using physical models has been shown to improve to student comprehension and engagement with the topic. We have developed a pre-lab activity that uses plastic building bricks and student "enzymes" to expose students to these concepts prior to conducting enzyme assays at the bench. Small groups of students take turns acting as an enzyme that catalyzes a hydrolysis reaction with increasing substrate concentration in the presence and absence of a competitive inhibitor. Students graph brick breaking rate data and make observations about the effect of changing parameters on key metrics. We conclude the activity with a class discussion on their observations. According to survey data, our students show an increase in the ability to answer conceptual and graphical questions correctly after completing the activity and corresponding material. Moreover, the majority of students thought that the activity was moderately or greatly helpful at increasing their understanding of key concepts. This kinesthetic active learning approach provides an engaging and fun way to introduce students to modeling enzyme kinetics and is adaptable to any class or laboratory setting.

Primary image: Breaking Bricks: A Hands-on Model of Enzyme Kinetics and Inhibition. Enzyme-catalyzed hydrolysis of a disaccharide into two monosaccharides is modeled by 2x2 plastic building bricks, with students’ hands representing the enzyme.

Students are frequently overwhelmed by the complexity of metabolic pathways and they think they have "learned" the pathway when they have memorized the individual reactions.  This laboratory lesson helps students to understand the significance of individual reactions in the pathways leading to methionine synthesis in the budding yeast, Saccharomyces cerevisiae.  Students appreciate that methionine is one of only two sulfur-containing amino acids, and students do not find it difficult to follow the "yellow" sulfur atom in the pathway. In the lesson, students use three different yeast met strains, each of which lacks a single gene involved in methionine synthesis.  Working in groups of three, students identify the missing MET gene in each of the three deletion strains by analyzing the abilities of the deletion strains to grow on several defined media in which methionine has been replaced with alternative sulfur sources. Students also determine the position of mutant genes in the pathway relative to sulfite reductase, using indicator media that reacts with sulfide, the product of the reaction catalyzed by sulfite reductase. For the analysis, students prepare serial dilutions of yeast cultures and spot the dilution series on agar plates. This lesson is part of a semester-long research investigation into the evolutionary conservation of the genes involved in methionine synthesis. The lesson can also be used as a stand-alone exercise that teaches students about biochemical pathways, while reinforcing basic microbiological techniques.

Reported misconceptions of enzyme-substrate interactions highlight the necessity for better, targeted instructional tools and assessments. A series of active learning activities with corresponding three-dimensional (3D) physical models were developed to target undergraduate biochemistry students’ conceptual understanding of space, electrostatic interactions, and stereochemistry in enzyme-substrate interactions. This lesson includes two activities utilizing physical models of elastase, chymotrypsin, and trypsin. These enzymes are widely taught in undergraduate biochemistry courses and are exceptional examples of a variety of enzyme paradigms. The Model Exploration activity guides students in an exploration of these models to connect conceptual and visual content. The Problem Solving activity uses two-dimensional representations of the physical models to further build student's understanding of enzyme-substrate interactions. These activities are implemented in two consecutive fifty-minute classes or alternatively combined for a seventy-five-minute class. These lessons are an inclusive, student-centered approach to teaching that enables students to confront misconceptions and promotes mastery of the material.

Primary image: Backbones and Surfaces and Substrates! Oh My! Undergraduate Biochemistry Students Working with the Serine Protease Model Set.

Developing student creativity and ability to develop a testable hypothesis represents a significant challenge in most laboratory courses. This lesson demonstrates how students use facets of molecular evolution and bioinformatics approaches involving protein sequence alignments (Clustal Omega, Uniprot) and 3D structure visualization (Pymol, JMol, Chimera), along with an analysis of pertinent background literature, to construct a novel hypothesis and develop a research proposal to explore their hypothesis. We have used this approach in a variety of institutional contexts (community college, research intensive university and primarily undergraduate institutions, PUIs ) as the first component in a protein-centric course-embedded undergraduate research experience (CURE) sequence. Built around the enzyme malate dehydrogenase, the sequence illustrates a variety of foundational concepts from the learning framework for Biochemistry and Molecular Biology. The lesson has three specific learning goals: i) find, use and present relevant primary literature, protein sequences, structures, and analyses resulting from the use of bioinformatics tools, ii) understand the various roles that non-covalent interactions may play in the structure and function of an enzyme. and iii) create/develop a testable and falsifiable hypothesis and propose appropriate experiments to interrogate the hypothesis. For each learning goal, we have developed specific assessment rubrics. Depending on the needs of the course, this approach builds to an in-class student presentation and/or a written research proposal. The module can be extended over several lecture and lab periods. Furthermore, the module lends itself to additional assessments including oral presentation, research proposal writing and the validated pre-post Experimental Design Ability Test (EDAT). Although presented in the context of course-based research on malate dehydrogenase, the approach and materials presented are readily adaptable to any protein of interest.

Primary image: Mind map of the hypothesis development.

Potential Activities for Biochem