TABLE 5.

Transport and related activities in various uncoupled mutants^a

Allele of mtlA	Transport			Phosphor- ylation (%)	Binding constant (nM)	Free intracellular mannitol	Uncoupling
	Activity (nmol/min/mg of total protein)	Km (μM)	Vmax (nmol/min/mg of total protein)
E. coli K-12
Wild type	9.5	4	82	100	40	No	No
E218A	11.5	5	25	110	—b	NT	Yes
E218V	≤0.1			12	No	NT	Yes
H256P	≤0.1			≤1	No	NT	Yes
K. pneumoniae
Wild type	3.1	1.2	33	100	NT	No	No
E218A	3.4	2.9	59	100	NT	No	Yes
H256P	≤0.1			14	NT	Yes	Yes
H256Y	NT	58.0	31	100	NT	No	Yes

^aCells of strain LGS324 transformed with plasmids carrying the complete mtlA gene either from E. coli K-12 on pGJ9 or from K. pneumoniae on pSOL300 in the wild-type and mutated forms were grown exponentially in minimal glycerol medium. Whole cells were used to determine the transport activity with 10 μM d-[¹⁴C]mannitol and to determine the apparent K_m values and the corresponding V_max values. Phosphorylation activities (100% corresponded to 278 nmol per min per mg of membrane protein) were determined with 500 μM d-[¹⁴C]mannitol or were estimated based on thin-layer chromatography tests as described in the text. The binding constants were determined by flow dialysis as described in Materials and Methods with membrane vesicles. The presence of free intracellular mannitol was determined by thin-layer chromatography (Fig. 2), and coupling was determined by measuring the growth on d-mannitol of transformants expressing the wild-type or mutated truncated IIC domains in the presence of DalD. NT, not tested.

^b—, see text.