5. Phenylketonuria (PKU) is an inherited disease which results from the lack of

Question

5. Phenylketonuria (PKU) is an inherited disease which results from the lack of the enzyme phenylalanine hydroxylase (PAH). The PAH enzyme catalyzes the first step in the degradation of Phe. In PKU patients, Phe accumulates and is eventually transaminated to phenylpyruvate. Excess phenylpyruvate accumulates in the blood and urine and has the effect of causing mental retardation if untreated. Screening programs identify PKU babies at birth, and treatment consists of a low Phe diet until maturation of the brain is completed. It is known that the PAH enzyme contains 451 residues and has a MW of 51.9 kDa. More than 60 different mutant genes giving rise to nonfunctional PAH proteins have been identified in PKU patients. a. In order to learn more about PAH, 3 isozymes were isolated from rat liver. Their MW ace. identical, but the pl values are 5.0, 5.6 and 6.1. A DEAE anion exchange cellulose column was used to separate the isozymes. What order of elution would you expect the isozymes to elute if a buffer at pH 5.8 is used along with a gradient of NaCI from 100 mM to 500 mM over 10 minutes? I would expect the elution to be 6.1,5.6, and lastly 5.0 b. Once purified, the investigators wanted to determine if Phe served as a regulator of PAH beyond being a substrate. Polyacrylamide gel (under denaturing and non-denaturing conditions) electrophoresis was performed and the results are shown in Fig. 1 below. Explain these results and your reasoning. (limit 200 words) (2 points) C. Next, kinetic studies were performed on PAH. A plot of velocity Phe concentration yielded a sigmoidally shaped curve. Explain these results and your reasoning. d. More kinetic data is shown in Fig. 2. interpret the data based on relative activity The effects of the hormones glucagon and insulin on PAH activity are shown in Fig. 3 below. Explain these results and your reasoning e. f. Tyr is not an essential amino acid in normal people, but it is essential in PKU patients. Give an explanation for this.

Explanation / Answer

a. The isoforms of PAH isozymes with pI less than pH 5.8 will carry negative charges. Therefore, PAH isoforms with pI 5.0 is highly negatively charged than the isoform with pI 5.6. The later will be eluted first in low salt elution buffer. Since the PAH isozyme with pI 5.0 are (highly negatively charged) binded to the DEAE anion exchange column (positive matrix), it will be eluted slowly in high salt elution buffer. The unbound proteins like PAH isoform with pI 6.1 will eluted first.  

Related Questions

Navigate

• Browse (All)
• Browse 5
• Subjects

---

---

Integrity-first tutoring: explanations and feedback only — we do not complete graded work. Learn more.