Thermal stability and CD analysis of rat tyrosine hydroxylase.
Tyrosine hydroxylase is the rate-limiting enzyme of catecholamine biosynthesis. It is a homotetramer made up of 56 kDa subunits. We examined the thermal stability of tyrosine hydroxylase purified from a rat pheochromocytoma cell line and investigated the relationship between enzyme activity and stability. Thermal stability was assessed by incubating the enzyme at an elevated temperature. Unfolding of the protein was followed by measuring the loss of circular dichroism (CD) at 220 nm. The CD loss was biphasic, with half-lives of 2 and 14 min at 55 degrees C in 100 mM potassium phosphate, pH 6.0. The rate of loss of enzyme activity paralleled the longer half-life under these conditions. This indicates that the structure of the active site is not appreciably change by the unfolding events corresponding to the first phase. Moreover, unfolding as assessed by the CD spectrum and activity was not reversible and did not exhibit a well-defined midpoint temperature or Tm. The thermal stability of the enzyme was altered by several factors that influence activity. The enzyme at pH 6.0 was less stable (t1/2 = 6.2 and 29 min) than the enzyme at pH 7.2 (a single t1/2 of 64 min). Phosphorylated tyrosine hydroxylase had shorter half-lives (t1/2 of 2 and 16 min) than the nonphosphorylated enzyme (t1/2 6.2 and 29 min) at pH 6.0, 50 degrees C, in 100 mM phosphate. Moderate changes in phosphate concentration had dramatic effects on enzyme stability. Decreasing the phosphate concentration from 50 to 10 mM (pH 6.0) increased the half-life from 2 and 23 min to greater than 120 min.(ABSTRACT TRUNCATED AT 250 WORDS)
Gahn
L G
LG
Department of Biochemistry and Molecular Biology, Louisiana State University Medical Center, New Orleans 70119.
Roskoski
R
R
D000818:Animals; D002942:Circular Dichroism; D004795:Enzyme Stability; D006863:Hydrogen-Ion Concentration; D016716:PC12 Cells; D010766:Phosphorylation; D000071228:Polyelectrolytes; D011108:Polymers / Q000494:pharmacology; D051381:Rats; D012492:Salts / Q000494:pharmacology; D013816:Thermodynamics; D014446:Tyrosine 3-Monooxygenase / Q000737:chemistry*
D000071228:Polyelectrolytes; D011108:Polymers; D012492:Salts; D014446:Tyrosine 3-Monooxygenase