Rapid quenching techniques

Each of these compounds, 53-56, was shown to be a very effective competitive inhibitor of the enzyme with respect to the fructose 1,6-diphosphate, whereas several other analogs, including acyclic structures, had no effect. These and other results suggest that the furanose form of the sugar diphosphate is the active form in the enzymatic reaction (105). More recent studies using rapid quenching techniques and C-nmr measurements have confirmed this hypothesis and indicate that the enzyme uses the a anomer 52 much more rapidly than the 3 anomer 50 and probably uses the a anomer exclusively (106). 

Raman scattering 192 Random mechanism 120 Rapid equilibrium mechanism 120 Rapid mixing techniques 133-136 Rapid quenching techniques 135-136... 

Most of these observations have since been verified. Phosphorylation by substrate has been shown to occur under acid conditions by using a stopped-flow technique (118, 165) as illustrated in Fig. 4. Under alkaline conditions the phosphoryl enzyme cannot normally be observed or isolated because the rate of dephosphorylation exceeds the maximum rate of phosphorylation (170). One interesting aspect is that the pH-rate profiles for phosphorylation and dephosphorylation are quite different, as is the case for E. coli alkaline phosphatase (171). Barman and Gut-freund studied the formation and breakdown of milk phosphoryl phosphatase using a rapid-quenching technique and concluded that dephosphorylation could not be rate limiting for the hydrolysis of p-nitrophenyl phosphate at pH 7 (S3). 

Valuable insights into how DNA polymerases process their substrates were obtained as a result of detailed kinetic studies of the enzymes. Benkovic and coworkers employed rapid quenching techniques to study the kinetics of transient intermediates in the reaction pathway of DNA polymerases [5]. Intensive studies revealed that E. coli DNA polymerase I follows an ordered sequential reaction pathway when promoting DNA synthesis. Important aspects of these results for DNA polymerase fidelity are conformational changes before and after the chemical step and the occurrence of different rate-limiting steps for insertion of canonical and non-canonical nucleotides. E. coli DNA polymerase I discriminates between canonical and non-canonical nucleotide insertion by formation of the chemical bond. Bond formation proceeds at a rate more than several thousand times slower when an incorrect dNTP is processed compared with canonical nucleotide insertion. 

Stokes et al. 47) reported similar conversions of oxygen in air to nitrogen oxides in a low-pressure RF plasma. After a run time of 2.5 hr, 0.4 ml of material was collected in liquid nitrogen traps, which on analysis showed a 2% conversion to nitric oxide. LaRoche 36) had previously demonstrated the beneficial effect of quenching the reaction products. In experiments with a low-pressure RF discharge, he obtained an increase in conversion to nitric oxide from 2 to 4% by a rapid quenching technique. 

Pre-steady-state stopped-flow and rapid quench techniques applied to Mo nitrogenase have provided powerful approaches to the study of this complex enzyme. These studies of Klebsiella pneumoniae Mo nitrogenase showed that a pre-steady-state burst in ATP hydrolysis accompanied electron transfer from the Fe protein to the MoFe protein, and that during the reduction of N2 an enzyme-bound dinitrogen hydride was formed, which under denaturing conditions could be trapped as hydrazine. A comprehensive model developed from a computer simulation of the kinetics of these reactions and the kinetics of the pre-steady-state rates of product formation (H2, NH3) led to the formulation of Scheme 1, the Thorneley and Lowe scheme (50) for nitrogenase function. 

Dunaway—Mariano s group has used rapid quench techniques to demonstrate the formation... 

---