Observation of a kinetic slow transition in monomeric glucokinase.

Rat liver glucokinase (EC 2.7.1.2) is a monomeric enzyme with positive cooperativity for glucose phosphorylation for which several kinetic mechanisms have been proposed. We have observed a slow kinetic transition when the enzyme is assayed in the presence of 30% glycerol. When the enzyme had been preincubated or stored in 50 mM glucose, the initially rapid activity decayed, via a first-order process, to a new steady-state velocity. The glucose-induced process is reversible since if the enzyme is preincubated without glucose, an initially low activity accelerates over minutes to the same steady-state velocity. This final velocity is independent of the preincubation conditions and is determined solely by the glucose and ATP concentrations in the assay. Possible artifacts which might cause nonlinear progress curves have been ruled out. The transition has a half-time of 2-10 min depending on glucose and ATP concentrations and temperature. In the steady-state kinetics, positive cooperativity occurs with glucose with a Hill coefficient (nH) = 1.3 at high ATP concentrations, approaching unity as the ATP concentration decreases. This pattern is similar to that seen in the linear velocities in the absence of glycerol. Similarly, negative cooperativity with MgATP is seen in the steady-state velocities at low glucose concentrations with the Hill coefficient approaching 1 as the glucose concentrations approach saturation. The initial velocity for enzyme preincubated in high glucose concentration was either Michaelis-Menten as a function of glucose at high MgATP concentration or heterogeneous (nH less than 1, negatively cooperative) at low MgATP concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Interconversions between different sulfhydryl-related kinetic states in glucokinase.

Rat liver glucokinase (EC 2.7.1.2) undergoes two distinct sulfhydryl-related reversible kinetic transitions. During normal assays in the presence of both substrates but without added reducing agents, the activity decays ("kappa" decay) over time to a new steady-state rate. The half-time for this decay is essentially constant at glucose levels from 2 to 200 mM and averages 6.2 +/- 2 min. Glucokinase in this kappa steady state displays an increased Km for glucose but has the same Vmax as normal, sulfhydryl-activated glucokinase. The kappa form does not itself exhibit kinetic cooperativity with glucose. In contrast, glucokinase incubated with neither glucose nor sulfhydryl reagents decays (mu decay) to a form whose Vmax is near zero. The t 1/2 for this transition is about 0.5 min at 0 or very low (0.5 mM) glucose concentrations. For both decays, incubations of enzyme with intermediate levels of reducing agents give steady-state mixtures of activated and either kappa and/or mu forms, depending on conditions during the decay. Enzyme at intermediate stages of the kappa decay displays an unchanged Vmax, intermediate (increased relative to activated enzyme) glucose S0.5 values, and diminished glucose cooperativity. In contrast, enzyme at intermediate steady-state mixtures of activated and mu forms has a normal glucose S0.5 and cooperativity but a diminished Vmax from the activated states. The enzyme at any stage of each decay may be fully reactivated by the addition of sulfhydryl reducing agents such as dithiothreitol, dithioerythritol, glutathione, or mercaptoethanol. A model is proposed to account for this complex behavior in glucokinase kinetics which proposes different enzymatic states (kappa and mu) locked in by sulfhydryl oxidation of different conformations dictated by glucose concentration. These sulfhydryl-related transitions may be important in regulation of glucokinase activity, since glucokinase is very sensitive (at least 20-fold differential activity) to concentrations of glutathione within the physiological range, perhaps allowing the normally variable glutathione levels or cytosolic redox potential to modify the rate of uptake and storage of blood glucose through control of glucokinase activity.

The active site of C3a anaphylatoxin.

The essential active site responsible for the inflammatory activities of C3a, an anaphylatoxin derived from the serum complement system, has been elucidated using C3a peptides synthesized by the solid phase method and assayed for their ability to contract guinea pig ileal tissue and to produce a wheal and flare response in human skin. The COOH-terminal C3a pentapeptide (Leu-Gly-Leu-Ala-Arg) common to rat, pig, and man shows vascular and smooth muscle activity as well as specificity similar to natural human C3a. The porcine C3a octapeptide is 3 times more active than the common pentapeptide, but the human octapeptide (Ala(70)-Ser-His-Leu(73)-Gly-Leu(75)-Ala-Arg(77) is 12 times more active than the pentapeptide. Replacement of the serine and histidine by alanine or acetylation of the NH2 terminus provides analogues with the same activity as the octapeptide. Thus, the increased activity of the human C3a octapeptide over the pentapeptide appears to be related to the backbone of residues 70 to 72 and is not due to the presence of the hydroxyl group of serine-71, the imidazole ring of histidine-72, or a positive charge at or near the NH2 terminus. Since the COOH-terminal tetrapeptide is 40 times less active than the pentapeptide, an adequate model of the essential active site of C3a anaphylatoxin is the common COOH-terminal pentapeptide region. A C3a active site analogue containing a COOH-terminal lysyl residue is devoid of ileal activity. In addition, the [alanine-73]pentapeptide is 9 times less active and the [alanine-75]pentapeptide is at least 70 times less active than the active site pentapeptide in the ileal assay. Thus, the hydrophobic side chains of leucine-73 and leucine-75 and the guanidinium group of arginine-77 are important for the contractile activity of the active site COOH-terminal pentapeptide of human C3a anaphylatoxin.