Biochemistry of a newly discovered (pretend) neurotransmitter

Vesicles containing neurotransmitters are synthesized in the cell body, or “soma” of the neuron and carried down microtubule tracks by motor proteins. For exocytosis to occur, these vesicles must fuse with the membrane of the axon terminal, releasing their contents into the cleft.

NTKA is a newly discovered excitatory neurotransmitter. After its release into the synaptic cleft, NTKA is cleaved into two components, NT and KA, by an enzyme known as NTKAse. The KA component diffuses away into the extracellular space, while the NT component is brought back into the presynaptic neuron via specific membrane-bound transport proteins. Inside the axon terminal, NT is covalently bonded to another KA group, forming a new NTKA molecule that can be reused as a neurotransmitter.

A new disease, with an autosomal recessive mode of inheritance, is characterized by NTKA deficiency. Autosomal recessive NTKA deficiency is caused by a hyperactive NTKAse enzyme, “$∆$‍NTKAse,” which binds NTKA with a much higher affinity than wild-type NTKAse does, quickening the depletion of NTKA from the synaptic cleft. Interestingly, $∆$‍NTKAse and NTKAse do not appear to have different rates of catalysis at saturating substrate, “${\text{k}}_{cat}$‍”.

Three patients volunteer for a study involving NTKA deficiency. Via in vitro genetic manipulation, researchers are able to produce a large quantity of NTKAse protein from each of these patients and characterize them biochemically. Figure $1$‍ shows a Lineweaver-Burke plot of NTKAse activity for three patients, each of whom has one or more relatives with NTKA deficiency. Patients $1$‍ and $2$‍ are of unknown genotype, while patient $3$‍ is homozygous recessive. The NTKAse activity of Patient $3$‍ was measured in the presence of a drug known to limit the activity of $∆$‍NTKAse.

A Lineweaver-Burke plot can be used to characterize an enzyme’s activity in different concentrations of substrate. The reciprocal of the enzyme’s rate of reaction, $1/V$‍, is plotted against the reciprocal of the substrate concentration, $1/[$‍S$]$‍. The enzyme’s maximum rate of reaction, ${\text{V}}_{max}$‍, and the enzyme’s binding affinity for substrate, ${\text{K}}_M$‍, can be extracted from the plot’s $y$‍- and $x$‍-intercepts, respectively. A low ${\text{K}}_M$‍ indicates high binding affinity, whereas a high ${\text{K}}_M$‍ indicates low binding affinity.

Figure 1: A Lineweaver-Burke plot was used to characterize the ${\text{V}}_{max}$‍ and ${\text{K}}_M$‍ values of NTKAse in three patients. The ${\text{V}}_{max}$‍ and ${\text{K}}_M$‍ values of each patient can be extracted by taking the inverses of the $y$‍- and $x$‍-intercepts, respectively. The third patient’s assay includes an inhibitor of $∆$‍NTKAse.