Crystallization and preliminary X-ray studies of Escherichia coli glycerol kinase

Abstract

Escherichia coli glycerol kinase, a major regulatory enzyme which catalyzes the reversible MgATP-dependent phosphorylation of glycerol has been crystallized by the hanging drop vapor diffusion method at room temperature. Three different crystal forms have been obtained in the presence of glycerol and appear to be suitable for X-ray crystallographic studies. Vapor diffusion against 55% ammonium sulfate and 1% β-octyl glucoside (pH 7.0) yields rhombohedral crystals with space group R32, a = b = 277.1 A ̊ , c = 78.7 A ̊ (hexagonal indexing) containing a dimer of M r 112,000 in the asymmetric unit ( V m = 2.64 A ̊ 3/dalton ). Vapor diffusion against sodium chloride in the presence of 10% (w/v) polyethylene glycol (pH 6.5 to 7.0) yields two different crystal forms, both with space group P2 1. The first form has a = 88.1 A ̊ , b = 99.3 A ̊ , c = 114.6 A ̊ , β = 119 ° , the second form has a = 92.5 A ̊ , b = 117.6 A ̊ , c = 108.3 A ̊ , β = 93.64 ° . Addition of ADP enhances growth of the monoclinic forms. These forms appear to contain an entire tetramer of M r 224,000 in the asymmetric unit and have V m values of 2.28 and 2.65 Å 3/dalton, respectively. All forms diffract to better than 3.0 Å resolution while the second monoclinic form diffracts to approximately 1.8 Å.