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Petur Orri Heidarsson  
  
 
 
 
 

 
 

Alkaline phosphatase

Single-amino acid mutations can alter the properties of the cold-adapted alkaline phosphatase we have worked with from a marine Vibrio species.

Generally, the catalytic efficiency (kcat/Km) of cold-adapted enzymes is several-times higher than for the homologous enzymes from mesophilic organisms.  The kcat is always increased when compared at 25°C, whereas Km may remain little changed or also increase.  Both effects can be related to a looser structural coherence, a trait believed to be advantageous when enzymes cool.

Thermal stability as monitored by spectroscopy (CD or fluorometry) or caloriometry is often reduced by cold-adaptation, pointing to global destabilization.  However, thermal tolerance of activity is often even lower, indicating local softness around the catalytic machinery.  Alkaline phosphatase (AP) from a Vibrio sp. has no disulfide bridge in contrast to the two present in most other APs.  This may invoke greater movement in the structure.  We have introduced four disulfide bridges that cause increased stability and higher Km values together with large decreases in kcat. Each bridge connects the only native cysteine near the active site to any of four engineered cysteines lying in adjacent positions on a nearby surface loop (Figure). 

Electron spin resonance has been used to confirm restricted mobility of the helix carrying the native cysteine-67. Several residues in the active site have been altered to imitate metal ligands observed in more thermostable APs.  As an example, the replacement of a Trp with a His (W274H), the residue 328 in E. coli AP, produced a more rigid and stable structure with much reduced catalytic efficiency (see Fig. in panel). 

It is inferred that an enhancement of structural mobility is created by reduction in internal weak interactions that gives cold-adapted enzymes greater catalytic power at low temperatures. This optimal balance between structural stability and structural flexibility proves quite sensitive to single residues replacements local to the active-site in Vibrio AP.

 Overall conclusions so far ...

• Our results support the notion that an optimal balance between global structural stability and local structural flexibility gives cold-adapted enzymes greater catalytic power at low temperatures.

• Mutations inside the active site can take part in producing cold-active properties.

• Restriction of mobility near the active site by Cys-Cys cross-linking lowered kcat but enhanced substrate binding (decreased Km).

• EPR was capable of giving information on local structural mobility.


 
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