Defense Date

6-18-2024

Graduation Date

Summer 8-10-2024

Availability

Immediate Access

Submission Type

dissertation

Degree Name

PhD

Department

Chemistry and Biochemistry

School

School of Science and Engineering

Committee Chair

David W. Seybert

Committee Member

Mihaela-Rita Mihailescu

Committee Member

Michael Cascio

Committee Member

Joseph McCormick

Keywords

macromolecular crowding, enzyme kinetics, lactate dehydrogenase, pkm2, cooperativity, substrate inhibition

Abstract

Macromolecules occupy up to 30% of a cell's volume, yet the in vitro study of biological processes is almost exclusively performed in dilute solutions that fail to approximate the crowded interior of a cell. A growing body of literature has identified that macromolecular crowding has significant effects on a number of biochemical processes, including enzymatic activity. However, changes in kinetic parameters with increased macromolecule concentrations have only been determined for enzymes that follow hyperbolic Michaelis-Menten kinetics, despite the fact that over 25% of enzymes deviate from this model. Here, we perform the first characterization of macromolecular crowding effects on enzymes that display non-hyperbolic kinetics by examining three enzymes that display substrate inhibition and/or cooperativity: two bacterial lactate dehydrogenases and a human pyruvate kinase isoform.

The D-lactate dehydrogenase from Lactobacillus delbrueckii subsp. lactis (Ldl-LDH) is non-cooperative and displays pyruvate substrate inhibition, while L-lactate dehydrogenase from Lacticaseibacillus casei (Lc-LDH) displays both pyruvate substrate inhibition and cooperativity. We find that macromolecular crowding has the opposite effect on substrate inhibition for these isozymes. Increased macromolecule concentrations up to just 10% (w/v) produce significant enhancement of pyruvate substrate inhibition for Ldl-LDH. This results in a reduction of the substrate concentration yielding maximal activity (Cmax) from 50% higher than the physiological pyruvate concentration to a value 25% lower than the physiological pyruvate concentration. In contrast, physiologically relevant concentrations of a model macromolecular crowder, the activator fructose 1,6-bisphosphate (FBP), or ATP each individually reduce or eliminate substrate inhibition in Lc-LDH. These results suggest that pyruvate substrate inhibition is likely operative in vivo for Ldl-LDH but not Lc-LDH; this distinction is otherwise lost when these enzymes are only studied in dilute solution.

To further probe the effects of macromolecular crowding on cooperativity, we also conducted experiments with the cooperative Lc-LDH in addition to human pyruvate kinase, muscle isoform 2 (hPKM2). Increased macromolecular crowding with both of these enzymes results in reduced cooperativity and activation by FBP, challenging previously assumed mechanisms of regulation based on assays performed in dilute solution. Further, studies have identified that hPKM2 activity is also regulated by several free amino acids, and we expand this knowledge by characterizing the time-dependent regulation of hPKM2 by serine or alanine. These experiments also lead to the characterization of a dilution-induced hPKM2 activity loss, highlighting how the results obtained from the in vitro study of hPKM2 are highly dependent on assay conditions and methodology.

Our results build upon previous macromolecular crowding literature to identify that macromolecular crowding effects on the substrate inhibition constant, Ki, are enzyme---and even isozyme---dependent. In contrast, we identify an initial trend of reduced cooperativity with increased macromolecule concentrations in both prokaryotic and eukaryotic systems. As cooperativity is commonly regarded as an important regulatory mechanism, this work underscores the need to study additional cooperative enzymes using physiological macromolecule concentrations to better understand regulatory processes in vivo.

Language

English

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