Enzyme Concentration And Incubation Time

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The objective of this experiment was to determine if N-acetyl-B-D-hexosaminidase isolated from rat testes exhibits Michaelis-Menten kinetics. This was determined by first deciding the optimal enzyme concentration and incubation time, 1.32*10-5 μM/mL and 60 mins, respectively. The enzyme was then reacted with varying concentrations of 4-nitrophenyl-N-acetyl-B-D-glucosamine enzyme substrate and a Michaelis-Menten plot was generated. Once it was determined that N-acetyl-B-D-hexosaminidase follows Michaelis-Menten kinetics by exhibiting a hyperbolic curve, a Lineweaver-Burk plot was generated and kinetic parameters Vmax, Km, Kcat, and Kcat/Km ratio were calculated. The parameters Vmax, Km, Kcat, and Kcat/Km ratio were determined to be 165 μm/mL/min, 0.145 mM, 2.08*105 s-1, and 1.43*106 mM-1 s-1, respectively. N-acetyl-B-D-hexosaminidase was proven to follow Michaelis-Menten kinetics, however, the calculated enzymatic parameters did not aligned with literature values.

INTRODUCTION

Enzyme kinetics is a field of study that allows for enzymes’ catalytic role within reactions to be quantified. Quantifying enzymes’ catalytic role provides a means to characterize enzymes through various lenses. Enzyme kinetics can be studied through the use of multiple kinetic parameters. Km, Vmax, Kcat, and the ratio of Kcat/Km can be determined to accurately quantify and measure aspects of an enzyme within a specific reaction. These parameters can further describe defining features of enzymes such
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